Methods and compositions for tissue regeneration

ABSTRACT

Disclosed is a composition comprising cells comprising keratinocytes or fibroblasts, or mixtures thereof, that secrete one or more biologically active molecules selected from the group consisting of GM-CSF, VEGF, KGF, bFGF, TGFβ, angiopoietin, EGF, IL-ID, IL- 6 , IL- 8 , TGFα, and TNFα and an extracellular matrix comprising alginate, wherein the cells are allogeneic and mitotically inactive.

This application is a continuation of co-pending U.S. application Ser.No. 12/255,481, filed Oct. 21, 2008, which is a continuation of U.S.patent application Ser. No. 10/526,853 filed Jan. 9, 2006 (now issued asU.S. Pat. No. 7,449,333), which is a U.S. national phase applicationunder 35 U.S.C. §371 of PCT Application No. PCT/US2003/027888 filed onSep. 5, 2003, which claims priority to U.S. patent application Ser. No.10/324,257 filed on Dec. 19, 2002 (now issued as U.S. Pat. No.7,144,729), which claims priority to U.S. Provisional Application No.60/408,565 filed on Sep. 6, 2002. The contents of all of theabove-referenced applications are incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to tissue regeneration, e.g.,the treatment of wounds using growth factor-, cytokine-, or angiogenicfactor-secreting cells admixed with a biological or syntheticextracellular matrix and/or attached or applied to a wound dressing orsolid nondegradable support matrix.

BACKGROUND OF THE INVENTION

Wounds (i.e., lacerations or openings) in mammalian tissue can result intissue disruption and coagulation of the microvasculature at the woundface. Repair of such tissue represents an orderly, controlled cellularresponse to injury. All soft tissue wounds, regardless of size, heal ina similar manner. The mechanisms of tissue growth and repair arebiologic systems wherein cellular proliferation and angiogenesis occurin the presence of an oxygen gradient. The sequential morphological andstructural changes, which occur during tissue repair have beencharacterized in great detail and have, in some instances, beenquantified. See Hunt, T. K., et al., “Coagulation and macrophagestimulation of angiogenesis and wound healing,” in The surgical wound,pp. 1-18, ed. F. Dineen & G. Hildrick-Smith (Lea & Febiger,Philadelphia: 1981).

Tissue regeneration in various organs, such as, e.g., the skin or theheart depends on connective tissue restoring blood supply and enablingresidual organ-specific cells such as keratinocytes or muscle cells toreestablish organ integrity. Thus, a relevant function of themesenchymal cells, i.e., the fibroblasts or, in addition, theendothelial cells of vasculature, is secretion of factors enhancing thehealing process, e.g., factors promoting formation of new blood vessels(angiogenesis) or factors promoting re-epithelialization byproliferating and migrating keratinocytes.

The cellular morphology of a wound consists of three distinct zones. Thecentral avascular wound space is oxygen deficient, acidotic andhypercarbic, and has high lactate levels. Adjacent to the wound space isa gradient zone of local ischemia, which is populated by dividingfibroblasts. Behind the leading zone is an area of active collagensynthesis characterized by mature fibroblasts and numerous newly formedcapillaries (i.e., neovascularization). While new blood vessel growth(angiogenesis) is necessary for the healing of wound tissue, angiogenicagents generally are unable to fulfill the long-felt need of providingthe additional biosynthetic effects of tissue repair. Despite the needfor more rapid healing of wounds (i.e., severe burns, surgicalincisions, lacerations and other trauma), to date there has been onlylimited success in accelerating wound healing with pharmacologicalagents.

The primary goal in the treatment of wounds is to achieve wound closure.Open cutaneous wounds represent one major category of wounds. Thiscategory includes acute surgical and traumatic, e.g., chronic ulcers,burn wounds, as well as chronic wounds such as neuropathic ulcers,pressure sores, arterial and venous (stasis) or mixed arterio-venousulcers, and diabetic ulcers. Open cutaneous wounds routinely heal by aprocess comprising six major components: i) inflammation, ii) fibroblastproliferation, blood vessel proliferation, iv) connective tissuesynthesis, v) epithelialization, and vi) wound contraction. Woundhealing is impaired when these components, either individually or as awhole, do not function properly. Numerous factors can affect woundhealing, including malnutrition, infection, pharmacological agents(e.g., cytotoxic drugs and corticosteroids), diabetes, and advanced age.See Hunt et al., in Current Surgical Diagnosis & Treatment (Way;Appleton & Lange), pp. 86-98 (1988).

Skin wounds that do not readily heal can cause the subject considerablephysical, emotional, and social distress as well as great financialexpense. See e.g., Richey et al., Annals of Plastic Surgery 23(2):159-65(1989). Indeed, wounds that fail to heal properly finally may requiremore or less aggressive surgical treatment, e.g., autologous skingrafting. A number of treatment modalities have been developed asscientists' basic understanding of wounds and wound healing mechanismshas progressed.

The most commonly used conventional modality to assist in cutaneouswound healing involves the use of wound dressings. In the 1960s, a majorbreakthrough in wound care occurred when it was discovered that woundhealing with a moist occlusive dressings was, generally speaking, moreeffective than the use of dry, non-occlusive dressings. See Winter,Nature 193:293-94 (1962). Today, numerous types of dressings areroutinely used, including films (e.g., polyurethane films),hydrocolloids (hydrophilic colloidal particles bound to polyurethanefoam), hydrogels (cross-linked polymers containing about at least 60%water), foams (hydrophilic or hydrophobic), calcium alginates (nonwovencomposites of fibers from calcium alginate), and cellophane (cellulosewith a plasticizer). See Kannon et al., Dermatol. Surg. 21:583-590(1995); Davies, Burns 10:94 (1983). Unfortunately, certain types ofwounds (e.g., diabetic ulcers, pressure sores) and the wounds of certainsubjects (e.g., recipients of exogenous corticosteroids) do not heal ina timely manner (or at all) with the use of such dressings.

Several pharmaceutical modalities have also been utilized in an attemptto improve wound healing. For example, treatment regimens involving zincsulfate have been utilized by some practitioners. However, the efficacyof these regimens has been primarily attributed to their reversal of theeffects of sub-normal serum zinc levels (e.g., decreased host resistanceand altered intracellular bactericidal activity). See Riley, Am. Fam.Physician 24:107 (1981). While other vitamin and mineral deficiencieshave also been associated with decreased wound healing (e.g.,deficiencies of vitamins A, C and D; and calcium, magnesium, copper, andiron), there is no strong evidence that increasing the serum levels ofthese substances above their normal levels actually enhances woundhealing. Thus, except in very limited circumstances, the promotion ofwound healing with these agents has met with little success.

What is needed is a safe, effective, and interactive means for enhancingthe healing of extensive and/or hard-to-heal wounds that can be usedwithout regard to the type of wound or the nature of the patientpopulation.

SUMMARY OF THE INVENTION

The present invention relates to the use of angiogenic or other growthfactors or cytokines expressed by human cells in unencapsulatedpreparations (mixed or combined with matrix material or syntheticbiocompatible substances) to be temporarily applied to wounds or defectsin skin or other tissues for the restoration of blood supplyingconnective tissue to enable organ-specific cells to reestablish organintegrity as well as to inhibit excessive scar formation.

In one aspect, the invention involves a cell preparation useful fortissue regeneration, e.g., for use in the treatment of skin wounds,containing one or more cell types that secrete one or more biologicallyactive substances, admixed with or applied to an extracellular matrix ormatrix material such that the admixture forms a viscous or polymerizedcell preparation. As used herein, the term “admixed” encompasses anymethods of combining, mixing, blending, joining etc. known to thoseskilled in the art. The cell types used in the cell preparation of theinvention are allogeneic, optionally mitotically inactivated, andselected from the group consisting of stromal, epithelial or organspecific, or blood-derived cells. For example, the cell types may bedifferentiated fibroblasts and keratinocytes. In other embodiments, thecell types may be selected from the group consisting of fibroblasts,keratinocytes (including outer root sheath cells), melanocytes,endothelial cells, pericytes, monocytes, lymphocytes (including plasmacells), thrombocytes, mast cells, adipocytes, muscle cells, hepatocytes,neurons, nerve or neuroglia cells, osteocytes, osteoblasts, cornealepithelial cells, chondrocytes, and/or adult or embryonic stem cells.

The main cell type of connective tissue is the fibroblast. Untilrecently, fibroblasts have been dealt with like homogenousnon-differentiating cell populations. However, the fibroblast cellsystem in various species, including man, is a stem cell system in whichthe fibroblasts terminally differentiate along seven stages, threecontaining mitotic and four including post-mitotic cells. See Bayreutheret al., Proc. Natl. Acad. Sci. USA 85:5112-16 (1988); Bayreuther et al.,J. Cell. Sci. Supple 10:115-30 (1988). In vitro induction of fibroblastdifferentiation may be performed by chemical or biological agents, suchas mitomycin C (Brenneisen et al., Exp. Cell. Res. 211:219-30 (1994)) orgrowth factors or cytokines (Hakenjos et al., Int. J. Radiat. Biol.76:503-09 (2000)) such as TGF beta 1, IL-1, IL-6, Interferon alpha. Invitro induction may also be accomplished by irradiation, e.g., withγ-rays; X-rays (Bumann et al., Strahlenther. Onkol. 171:35-41 (1995); UVlight (Rodemann et al., Exp. Cell. Res. 180:84-93 (1989); or physicalexposure to electromagnetic fields (Thumm et al., Radiat. Environ.Biophys. 38:195-99 (1999). Moreover, induction of differentiation mayalso be accomplished by culture conditions such as serum starvation,contact inhibition, or the addition of Mitomycin C. See Palka et al.,Folia Histochem. Cytobiol. 34:121-27 (1996).

To date, the function/biological properties of differentiatedfibroblasts have been poorly studied. The pattern of polypeptideexpression and secretion, however, varies from mitotic to post-mitoticstages. The respective polypeptides are still being analyzed. See, e.g.,Francz, Eur. J. Cell. Biol. 60:337-45 (1993).

In some embodiments, the biologically active molecule is at least oneangiogenic factor, at least one growth/cytokine factor, or a combinationof at least one angiogenic factor and at least one growth/cytokinefactor. Examples of suitable biologically active molecules include, butare not limited to, epidermal growth factor-growth factor family (EGF);transforming growth factor alpha (TGF alpha); hepatocyte growth factor(HGF/SF); Heparin-binding epidermal growth factor (EGF); basicfibroblast growth factor (bFGF); acidic fibroblast growth factor (aFGF);other fibroblast growth factors (FGF); keratinocyte growth factor (KGF);transforming growth factors (TGF) β1 and β2; transforming growth factor(TGF) β3; platelet derived growth factor (PDGF); vascular endothelialgrowth factor (VEGF); tumor necrosis factor (TNF); interleukin-1 (IL-1)and interleukin-6 (IL-6); other interleukin/cytokine family members;insulin-like growth factor I (IGF-1); colony-stimulating factor 1(CSF-1); and granulocyte macrophage colony stimulating factor (GM-CSF).Those skilled in the art will recognize that additional biologicallyactive molecules can also be used in the methods and compositions of theinvention.

In various embodiments, the extracellular matrix or matrix material usedcan be collagen, alginate, alginate beads, agarose, fibrin, fibrin glue,fibrinogen, blood plasma fibrin beads, whole plasma or componentsthereof, laminins, fibronectins, proteoglycans, HSP, chitosan, heparin,and/or other synthetic polymer or polymer scaffolds and solid supportmaterials, such as wound dressings, that could hold or adhere to cells.In preferred embodiments, the extracellular matrix or matrix material isselected from the group consisting of fibrin, fibrin glue, fibrinogen,fibrin beads, and other synthetic polymer or polymer scaffolds or wounddressing materials.

In a further embodiment, the cell types are mitotically inactivated,e.g., induced to various stages of differentiation. For example, thismitotic inactivation can be accomplished by the administration ofmitomycin C or other chemically-based mitotic inhibitors, irradiationwith γ-Rays, irradiation with X-Rays, and/or irradiation with UV light.

In another embodiment, the cell types are immortalized using at leastone gene/polypeptide selected from the group consisting of the 12S and13S products of the adenovirus E1A genes, hTERT, SV40 small T antigen,SV40 large T antigen, papilloma viruses E6 and E7, the Epstein-BarrVirus (EBV), Epstein-Barr nuclear antigen-2 (EBNA2), human T-cellleukemia virus-1 (HTLV-1), HTLV-1 tax, Herpesvirus saimiri (HVS), mutantp53, myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src, c-fgr, myb, c-myc,n-myc, and Mdm2.

In still further embodiments, the cell types are genetically engineeredto secrete one or more biologically active molecules, such as at leastone angiogenic factor, at least one growth/cytokine factor, or acombination of at least one angiogenic factor and at least onegrowth/cytokine factor. This secretion may be constitutive, or it may becontrolled by gene switching.

In various other embodiments, the invention also provides methods oftreating tissue defects or wounds by administering the cell preparationsaccording to the invention to a wound site on a patient in need of woundtreatment. The cell preparation of the invention can be administeredlocally (i.e. as a paste) to a wound site on a patient to temporarilyinduce tissue regeneration by biological interaction with surroundingtissues. Alternatively, the cell preparation of the invention can beadministered by spraying the components on a wound site on a patient totemporarily induce tissue regeneration by biological interaction withsurrounding tissues. The sprayed cell preparation may result in theformation of a matrix on the wound site.

In another aspect, the invention involves a method of manufacturing acell preparation for tissue regeneration by providing a firstcomposition containing one or more cells types that secrete one or morebiologically active molecules admixed with thrombin, wherein the cellstypes are allogeneic, optionally mitotically inactivated, and selectedfrom the group consisting of stromal, epithelial/organ specific, andblood derived cells. The first composition is then combined with asecond composition containing an extracellular matrix or matrix materialcontaining fibrinogen, wherein the combination of the first and secondcompositions forms a viscous cell past suitable for tissue regeneration.The cell types employed in the cell preparation may naturally secretethe one or more biologically active molecules, or they may begenetically engineered to secrete the one or more biologically activemolecules.

In various embodiments, these cell types are differentiated fibroblastsand keratinocytes, and the biologically active molecule is at least oneangiogenic factor, at least one growth/cytokine factor, or a combinationof at least one angiogenic factor and at least one growth/cytokinefactor. In other embodiments, the cell types may be mitoticallyinactivated by administration of mitomycin C or other chemically-basedmitotic inhibitors, irradiation with γ-Rays, irradiation with X-Rays, orirradiation with UV light.

In other embodiments, the cell types are immortalized using at least onegene/polypeptide selected from the group consisting of the 12S and 13Sproducts of the adenovirus E1A genes, hTERT, SV40 small T antigen, SV40large T antigen, papilloma viruses E6 and E7, the Epstein-Bar Virus(EBV), Epstein-Barr nuclear antigen-2 (EBNA2), human T-cell leukemiavirus-1 (FITLY-1), HTLV-1 tax, Herpesvirus saimiri (HVS), mutant p53,myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src, c-fgr, myb, c-myc, n-myc, andMdm2.

In another aspect, this invention provides a kit for the preparation ofa cell preparation for tissue regeneration. This kit contains a firstcomponent containing an extracellular matrix or matrix material and asecond component containing one or more cell types that secrete abiologically active molecule, such as, at least one angiogenic factor,at least one growth/cytokine factor, or a combination thereof. Thesecell types are allogeneic, mitotically active or inactivated, andselected from the group consisting of stromal, epithelial/organ specificand blood derived cells. For example, these cell types may bedifferentiated fibroblasts and keratinocytes. The resulting cellpreparation can be in the form of a paste. In various embodiments, theextracellular matrix or matrix material can be fibrin, fibrin glue,fibrinogen, fibrin beads, and other synthetic polymer or polymerscaffolds or wound dressing materials.

Mitotic inactivation of the cells can be accomplished by administrationof mitomycin C or other chemically-based mitotic inhibitors, irradiationwith γ-Rays, irradiation with X-Rays, or irradiation with UV light. Invarious aspects, the cell types are immortalized using at least one ofthe following: the 12S and 13S products of the adenovirus E1A genes,hTERT, SV40 small T antigen, SV40 large T antigen, papilloma viruses E6and E7, the Epstein-Barr Virus (EBV), Epstein-Barr nuclear antigen-2(EBNA2), human T-cell leukemia virus-1 (HTLV-1), HTLV-1 tax, Herpesvirussaimiri (HVS), mutant p53, myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src,c-fgr, myb, c-myc, n-myc, and Mdm2.

The cell types may naturally secrete the one or more biologically activemolecules or they may be genetically engineered to secrete an exogenouslevel of the one or more biologically active molecules. Secretion may becontrolled by gene switching or it may be constitutive. In oneembodiment, the first component contains fibrinogen. In anotherembodiment, the first component contains fibrinogen and the secondcomponent contains from about 1×10³ cells/μl to about 50×10³ cells/μl.The second component also contains thrombin and can optionally contain acryoprotectant such as a 10% glycerol solution, a 15% glycerol solution,and a 15% glycerol and 5% human serum albumin solution.

In another aspect, the invention provides methods for using the kits ofthe invention to prepare a cell preparation for tissue regeneration.This method involves administering the first component to a wound siteon a patient in need of treatment and combining the second componentwith the first component on the would site, wherein the combination ofthe first and second components forms a cell preparation suitable fortissue regeneration. In one embodiment, the first and second componentsare topically administered to the wound site on the patient. In anotherembodiment, the first and second components are sprayed onto the woundsite. The components can be sprayed such that they are combined on thewound or such that they are combined in the air before reaching thewound.

In yet another aspect, the invention provides methods of administering acell preparation for tissue regeneration to a wound site on a patient inneed of treatment. This method involves the steps of providing a firstcomponent containing an extracellular matrix or matrix materialcontaining fibrinogen; providing a second component containing fromabout 1×10³ cells/μl to about 50×10³ cells/μl and thrombin, wherein thecells secrete one or more biologically active molecules, are allogeneic,mitotically active or inactivated, and selected from the groupconsisting of stromal, epithelia/organ specific, and blood-derivedcells; combining the first and second components to form a cellpreparation suitable for tissue regeneration; and administering the cellpreparation to the wound site.

In one embodiment, the first and second components are topically appliedto the wound site. The first component can be applied to the wound sitebefore or after the second component is applied. In another embodiment,the first and second components are sprayed on the wound site.Preferably, the first component is sprayed on the wound site before thesecond component is sprayed on the wound site. The first and secondcomponents may be combined on the wound site or they may be combinedbefore reaching the wound site.

These cell types may be differentiated or undifferentiated fibroblastsand keratinocytes. The resulting cell preparation can be in the form ofa paste. In various embodiments, the extracellular matrix or matrixmaterial can be fibrin, fibrin glue, fibrinogen, fibrin beads, and othersynthetic polymer or polymer scaffolds or wound dressing materials.Mitotic inactivation of the cells can be accomplished by administrationof mitomycin C or other chemically-based mitotic inhibitors, irradiationwith γ-Rays, irradiation with X-Rays, or irradiation with UV light. Invarious embodiments, the cell types are immortalized using at least oneof the following: the 12S and 13S products of the adenovirus E1A genes,hTERT, SV40 small T antigen, SV40 large T antigen, papilloma viruses E6and E7, the Epstein-Barr Virus (EBV), Epstein-Barr nuclear antigen-2(EBNA2), human T-cell leukemia virus-1 (HTLV-1), HTLV-1 tax, Herpesvirussaimiri (HVS), mutant p53, myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src,c-fgr, myb, c-myc, n-myc, and Mdm2.

The cell types may naturally secrete the one or more biologically activemolecules or they may be genetically engineered to secrete an exogenouslevel of the one or more biologically active molecules. Secretion may becontrolled by gene switching or it may be constitutive. In oneembodiment, the second component can optionally contain a cryoprotectantincluding, but not limited to, a 10% glycerol solution, a 15% glycerolsolution, and a 15% glycerol and 5% human serum albumin solution.

In another aspect, the invention involves a cell preparation for tissueregeneration containing an extracellular matrix or matrix materialcontaining fibrinogen admixed with a second component containing fromabout 1×10³ cells/μl to about 50×10³ cells/μl and thrombin, wherein thecells secrete one or more biologically active molecules (such as atleast one angiogenic factor, at least one growth/cytokine factor, orcombinations thereof) are allogeneic, mitotically inactivated, andselected from the group consisting of stromal, epithelia/organ specific,and blood-derived cells.

These cell types may be differentiated fibroblasts and keratinocytes.The resulting cell preparation can be in the form of a paste. In variousembodiments, the extracellular matrix or matrix material can be fibrin,fibrin glue, fibrinogen, fibrin beads, and other synthetic polymer orpolymer scaffolds or wound dressing materials. Mitotic inactivation ofthe cells can be accomplished by administration of mitomycin C or otherchemically-based mitotic inhibitors, irradiation with γ-Rays,irradiation with X-Rays, or irradiation with UV light. In variousembodiments, the cell types are immortalized using at least one of thefollowing: the 12S and 13S products of the adenovirus E1A genes, hTERT,SV40 small T antigen, SV40 large T antigen, papilloma viruses E6 and E7,the Epstein-Barr Virus (EBV), Epstein-Barr nuclear antigen-2 (EBNA2),human T-cell leukemia virus-1 (HTLV-1), HTLV-1 tax, Herpesvirus saimiri(HVS), mutant p53, myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src, c-fgr,myb, c-myc, n-myc, and Mdm2.

The cell types may naturally secrete the one or more biologically activemolecules or they may be genetically engineered to secrete an exogenouslevel of the one or more biologically active molecules. Secretion may becontrolled by gene switching or it may be constitutive. In oneembodiment, the second component can optionally contain a cryoprotectantincluding, but not limited to a 10% glycerol solution, a 15% glycerolsolution, and a 15% glycerol and 5% human serum albumin solution.

The invention also provides methods of using such cell preparations byproviding the first and second components, combining the first andsecond components and administering the resulting cell preparation tothe wound site. In various embodiments, the components can be topicallyadministered to the wound site on the patient or they can be sprayedonto the wound site. When spray administered, the first and secondcomponents can be combined on the wound site or they can be combinedbefore reaching the wound site.

In another aspect, the first and second components of the kits of theinvention is cryopreserved prior to shipping and subsequently thawedprior to use. Each component may be contained in a separate vial havinga removable screw cap, wherein the vial is sterile and is made of amaterial resistant to low temperatures and wherein the removable lid canbe replaced with a spray pump following thawing of the first and secondcomponents prior to use. In one embodiment, the spray pump delivers avolume of approximately 130 μl per spray. Suitable materials resistantto low temperatures include, but are not limited to, glass,polypropylene, polyethylene, and ethylene vinyl acetate (EVA). In someembodiments, each vial may have a wall thickness of approximately 0.8 mland may hold a working volume of approximately 2 ml of the first andsecond components. In another embodiment, the vials are sealed within apouch or container prior to cryopreservation, wherein the pouch orcontainer is fabricated of a material capable of withstandingtemperatures ranging from −80° C. to −160° C. and wherein the pouch orcontainer protects the first and second components from contaminationduring cryopreservation, storage, and subsequent thawing. Preferable,the pouch is waterproof and has a high barrier performance.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram showing the results of a BrdU cell proliferationanalysis of human primary fibroblasts following 15 and 30 days inculture after gamma irradiation.

FIG. 2 is a histogram showing adherence of human primary fibroblasts toa petri dish following gamma irradiation treatments.

FIG. 3 is a table showing the results of testing for optimal dilutionsof thrombin and fibrinogen for the formulation of a spray applied fibringlue matrix.

FIG. 4 is a histogram comparing the quantity of growth factors secretedby cell matrices when different doses of cells are sprayed intoindividual wells of a 24 well culture dish. The figure also shows growthfactor secretion quantities when the fibrin matrix containing cells isprepared by simple pipetting (non-sprayed administration) of thefibrinogen and the thrombin+cell suspensions together.

FIGS. 5 a and 5 b are histograms demonstrating that mixing sprayedkeratinocytes and fibroblasts at different ratios, while maintaining aconstant total number of cells, gives rise to variable growth factorsecretion characteristics.

FIGS. 6 a and 6 b are pictures of a novel vial used for delivering andadministering the cell preparation of the invention. FIG. 6 a is a crosssection of the vial. FIG. 6 b is a three-dimensional drawing of theoutside of the vial, which is designed to hold a working volume of 2 mlof the component.

FIG. 7 is a histogram comparing growth factor secretion by cells storedfor one week at −160° C. versus cells stored at −80° C. when using a 10%glycerol solution as a cryoprotectant.

FIG. 8 is a histogram comparing growth factor secretion by cells storedfor one week at −80° C. in 10% glycerol versus 15% glycerol.

FIG. 9 is a histogram comparing growth factor secretion by cells storedfor one week at −80° C. in 15% glycerol versus 15% glycerol +5% HSA.

FIG. 10 is a histogram showing the secretion of the growth factorsGM-CSF and VEGF by cell preparations manufactured at differentproductions following either one, four, eight, or twelve weeks ofstorage at −80° C. In this experiment, the cell preparation componentswere frozen using 15% glycerol+5% human serum albumin as thecryoprotectant.

FIG. 11 is a histogram showing growth factor secretion by frozen cellpreparations stored frozen for one week at −80° C. Results for variouskeratinocyte:fibroblast ratios and number of cells are shown.

DETAILED DESCRIPTION OF THE INVENTION

Wound healing is a complex process involving soluble mediators, bloodcells, extracellular matrix, and parenchymal cells. Wound healing hasthree phases—the inflammation phase, the tissue formation phase, and thetissue remodeling phase. These phases may overlap in time.

Generally, an injury to tissue disrupts blood vessels and leads toextravasation of blood constituents. Blood clotting helps to reestablishhemostasis and provides a provisional extracellular matrix for cellmigration to the wound. At the wound site, platelets (thrombocytes)facilitate the formation of a hemostatic plug and also secrete severalmediators of the wound healing process. These mediators include, forexample, molecules such as platelet-derived growth factor that attractand activate monocytes and fibroblasts.

Soon after the injury, neutrophils infiltrate the wound and clean thewound of foreign particles and bacteria. The neutrophils are thenextruded with the eschar or undergo phagocytosis by macrophages.Monocytes also infiltrate the wound in response to specificchemoattractants, such as fragments of extracellular matrix proteins,transforming growth factors β, the monocyte chemoattractant protein 1,and subsequently become activated macrophages. These activatedmacrophages release growth factors such as platelet-derived growthfactor and vascular endothelial growth factor, which initiate theformation of granulation tissue. Macrophages bind through their integrinreceptors to proteins in the extracellular matrix. This bindingstimulates macrophage phagocytosis of any microorganisms as well as offragments of extracellular matrix.

Monocytes, stimulated by adherence to the extracellular matrix, alsoundergo metamorphosis into inflammatory macrophages. This adherence tothe extracellular matrix induces monocytes and macrophages to expresscolony-stimulating factor 1, tumor necrosis factor β, and plateletderived growth factor. Other important cytokines expressed by monocytesand macrophages are transforming growth factor α, interleukin-1,transforming growth factors β1-3, and insulin-like growth factor 1. Themonocyte- and macrophage-derived growth factors are thought necessaryfor the initiation and propagation of new tissue formation in wounds.

Within hours after the injury, reepithelialization of wounds begins.Keratinocytes from the wound edges as well as from residual skinappendages such as hair follicles undergo marked changes in phenotype,including retraction of intracellular tonofilaments, dissolution of mostintercellular desmosomes, and formation of peripheral cytoplasmic actinfilaments. Furthermore, the hemidesmosomal links between thekeratinocytes and the epidermal basement membrane dissolves, allowingthe movement of keratinocytes.

Within a few days post injury, the keratinocytes at the margin of thewound begin to proliferate behind the migrating cells. As thisreepithelialization occurs, the basement-membrane proteins reappear inan ordered sequence from the margin of the wound outward. Keratinocytesthen revert to their normal phenotype and attach themselves to thereestablished basement membrane and underlying dermis.

Within about four days post injury, new stroma begins to infiltrate thewound. Concomitantly, macrophages, fibroblasts, and blood vessels alsoinfiltrate the wound. Macrophages provide a source of growth factorsthat stimulate fibroplasia and angiogenesis. The fibroblasts produce thenew extracellular matrix that supports cell in-growth. The new bloodvessels carry oxygen and nutrients that sustain the cells.

Growth factors, particularly platelet-derived growth factor andtransforming growth factor β1, are thought to stimulate fibroblasts ofthe tissue around the wound to proliferate. In fact, platelet-derivedgrowth factor has been shown to accelerate the healing of chronicpressure sores and diabetic ulcers. Basic fibroblast growth factor hasalso been used with some success to treat chronic pressure sores.

However, there are many factors that can lead to abnormal wound healing.One example occurs with diabetic ulcers. Typically, diabetic ulcersexhibit multiple biochemical pathologies that can lead to impairedhealing. These ulcers occur in patients who cannot sense and relievecutaneous pressure due to some type of diabetic neuropathy. Frequently,diabetic ulcers become infected because of impaired granulocyticfunction and chemotaxis. Patients with diabetic ulcers also experienceinflammation, impaired neovascularization, decreased synthesis ofcollagen, increased levels of proteinases, and defective macrophagefunction.

Overall clinical experience using isolated, e.g., recombinant, growthfactors and other mediators to accelerate wound healing has not met withgreat success, perhaps because wound repair is the result of a complexset of interactions between soluble factors, formed blood elements,extracellular matrix, and cells. Combining various growth factors atcarefully controlled intervals may promote more effective wound healing.

The present invention provides stromal, epithelial, and blood-derivedcells, including, but not limited to, fibroblasts, keratinocytesincluding follicular outer root sheath cells, endothelial cells,pericytes, monocytes, lymphocytes including plasma cells, thrombocytes,mast cells, adipocytes, muscle cells, hepatocytes, nerve and neurogliacells, osteocytes, osteoblasts, corneal epithelial cells, andchondrocytes that are admixed with either a synthetic or naturalextracellular matrix (“ECM”) to form a cell preparation that can be usedto improve tissue granulation during wound healing. In one embodiment,the cells may deliver endogenous angiogenic factors or other growthfactors. In another embodiment, the cells can be genetically engineeredto produce exogenous amounts of the desired factor. Preferably, thecells are allogeneic. Those skilled in the art will recognize that thecells employed in the methods and preparations of the invention mayinclude, but are not limited to, living cells, mitotically inactivatedcells, mitotically activated cells, metabolically inactive cells,lyophilized cells and/or nonliving cells.

In particular, fibroblasts and keratinocytes have been shown to play animportant role in cutaneous wound healing. These roles includestimulating cell migration and proliferation, stimulating extracellularmatrix production, producing growth factors and cytokines, stimulatingangiogenesis, and releasing proteases which dissolve non-viable tissueand the fibrin barrier.

Wound healing may be promoted by use of growth and/or angiogenicfactors. For example, one suitable wound healing preparation consists oftwo cryopreserved components, i.e., fibrinogen and growth-arrested,allogeneic human fibroblasts and keratinocytes suspended in thrombin.After thawing, these components are sprayed sequentially on the woundbed to form a thin fibrin matrix containing two types of living, but notproliferating skin-derived cells, which, for several days, interactivelyproduce growth and angiogenic factors relevant for wound healing (e.g.VEGF, GM-CSF, bFGF, KGF).

The cells may be either immortalized or primary cell cultures. Cells maybe immortalized by any method known to those skilled in the art. Acommon approach to lengthening the lifespan of a cell is to transfer avirus or a plasmid that contains one or more immortalizing genes. Cellimmortalization increases the lifespan of a cell, and the resulting cellline is capable of being passaged many more times than the originalcells. Immortalizing genes are well known in the art. See, e.g.,Katakura et al., Methods Cell Biol. 57: 69-91 (1998). Immortalizingproteins or polypeptides include, but are not limited to, the 12S and13S products of the adenovirus E1A genes, SV40 small and large Tantigens, papilloma viruses E6 and E7, the Epstein-Barr Virus (EBV),Epstein-Barr nuclear antigen-2 (EBNA2), human T-cell leukemia virus-1(HTLV-1), HTLV-1 tax, Herpesvirus Saimiri (HVS), mutant p53, and theproteins from oncogenes such as myc, c-jun, c-ras, c-Ha-ras, h-ras,v-src, c-fgr, myb, c-myc, n-myc, and Mdm2. Additionally, cells maybecome spontaneously immortalized. A preferred immortalization strategyis by transfer of the gene encoding telomerase reverse transcriptase(TERT) into the cell such that TERT was either stably or transientlyexpressed thereby resulting in the expression of telomerase activity.Telomerase activity, when expressed in normal somatic cells, can lead toelongation of the chromosome tips or protective caps, called telomeres,thereby resulting in the ability of the telomerase expressing cells tobecome immortalized without becoming transformed (See Jiang, et al.,Nature Genetics 21:111-14 (1999) and Morales, et al., Nature Genetics21:115-18 (1999)).

Telomeres are specialized structures at the ends of eukaryoticchromosomes and that appear to function in chromosome stabilization,positioning, and replication. See Blackburn & Szostak, 53 Ann. Rev.Biochem. 163-194 (1984); Zakian, 23 Ann. Rev. Genetics 579-604 (1989);Blackburn, 350 Nature 569-573 (1991). In all vertebrates, telomeresconsist of hundreds to thousands of tandem repeats of the 5′-TTAGGG-3′sequence and associated proteins. See Blackburn, 350 Nature 569-573(1991); Moyzis et al., 85 Proc. Natl. Acad. Sci. 6622-6626 (1988).Southern blot analysis of chromosome terminal restriction fragments(TRF) provides the composite lengths of all telomeres in a cellpopulation. See Harley at al., 3445 Nature 458-460 (1990); Allsopp atal., 89 Proc. Natl. Acad. Sci. USA 10114-10118 (1992); Vaziri et al., 52Am. J. Human Genetics 661-667 (1993). In all normal somatic cellsexamined to date, TRF analysis has shown that the chromosomes lose about50-200 nucleotides of telomeric sequence per cell division, consistentwith the inability of DNA polymerase to replicate linear DNA to theends. See Watson, 239 Nature New Biology 197-201 (1972).

This shortening of telomeres has been proposed to be the mitotic clockby which cells count their divisions (see Harley, 256 Mut. Res. 271-282(1991)), and sufficiently short telomeres may be the signal forreplicative senescence in normal cells. See Hastie et al., 346 Nature866-868 (1990); Lindsey et al., 256 Mut. Res. 45-48 (1991); Wright &Shay, 8 Trends Genetics 193-197 (1992). In contrast, the vast majorityof immortal cells examined to date show no net loss of telomere lengthor sequence with cell divisions, suggesting that maintenance oftelomeres is required for cells to escape from replicative senescenceand proliferate indefinitely. See Counter et al., 11 EMBO 1921-1929(1992); Counter et al., 91 Proc. Natl. Acad. Sci. USA 2900-2940, 1994).

Telomerase, a unique ribonucleoprotein DNA polymerase, is the onlyenzyme known to synthesize telomeric DNA at chromosomal ends using as atemplate a sequence contained within the RNA component of the enzyme.See Greider & Blackburn, 43 Cell 405-413 (1985); Greider & Blackburn,337 Nature 331-337 (1989); Yu et al., 344 Nature 126-132 (1990);Blackburn, 61 Ann. Rev. Biochem. 113-129 (1992). With regard to humancells and tissues, telomerase activity has been identified in immortalcell lines and in ovarian carcinoma but has not been detected in mortalcell strains or in normal non-germline tissues. See Morin, 59 Cell521-529, 1989. Together with TRF analysis, these results suggesttelomerase activity is directly involved in telomere maintenance,linking this enzyme to cell immortality.

Expression of the human telomerase catalytic component (hTERT) hasrecently been studied in human somatic cells. See Jiang, et al., 21Nature Genetics 111-114 (1999). Telomerase expression in normal somaticcells did not appear to induce changes associated with a malignantphenotype such as abnormal growth control or oncogenic transformation.The absence of cancer-associated changes was also reported in humanfibroblasts immortalized with telomerase. See Morales, et al., 21 NatureGenetics 115-118 (1999). It was demonstrated that the introduction oftelomerase into normal human somatic cells does not lead to growthtransformation, does not bypass cell-cycle induced checkpoint controlsand does not lead to genomic instability of these cells. Methods fordetecting telomerase activity, as well as for identifying compounds orpolypeptides that regulate or affect telomerase activity, together withmethods for therapy or diagnosis of cellular senescence andimmortalization by controlling or measuring telomere length andtelomerase activity, have also been described (see PCT Internationalpatent application WO 93/23572). The identification of compoundsaffecting telomerase activity provides important benefits to efforts attreating human disease.

The cells according to the invention can also be genetically engineeredto produce one or more of biologically active molecules such that themolecules are constitutively secreted from the cells. By “constitutivelysecreted” is meant that the desired biologically active molecule iscontinuously expressed by the cells or that the gene is continuallyexpressed. Alternatively, the cells can be genetically engineered suchthat their expression is controlled by gene switching.

As used herein, the terms “gene switch” and “gene switching” refer tomethods of regulating gene expression. Specifically, expression of aprotein encoded by a gene is controlled by the interaction of certainregulatory proteins, known as DNA-binding proteins, with a regionlocated upstream of the gene. Within the promoter region, there arelocated several operator regions which contains a specificoligonucleotide sequence to which these DNA-binding proteinsspecifically bind. These proteins can lead either to activation orrepression of gene expression. Thus, they control the regulatedexpression of genes.

The regulator protein is encoded by a regulatory gene located elsewhereon the chromosome. The interaction of regulator and operator is affectedby the presence or absence of particular chemical factors (inducers).Thus, in normal circumstances the regulator is expressed, therebybinding the operator and inhibiting expression of the gene, until a needfor the particular protein encoded by the gene is indicated by theappearance in the environment of a specific inducer which interacts withthe regulator to inhibit binding to the operator, thus allowingexpression of the gene.

For example, an enzyme, which acts upon a sugar molecule is not requiredunless that sugar is present and, therefore, in the absence of thesugar, the regulatory gene expresses the regulator protein, which bindsthe gene operator and inhibits expression of the enzyme. The sugaritself acts as the inducer, which then interacts with the regulator toprevent its binding to the operator thus allowing expression of theenzyme. Digestion of the sugar by the enzyme removes it from theenvironment allowing the regulator to return to its normal mode and actnormally to inactivate enzyme expression.

Such a regulatory mechanism can be viewed as a switching arrangementwhich switches gene expression on and off as dictated by the chemicalcontent of the environment. Gene switching systems of the type describedare best known in bacteria and many of the proteins and their target DNAbinding sites are known in considerable detail. The regulator proteinsusually bind as dimers to operators, which exhibit a two-fold symmetry.The specificity of the regulator/promoter interaction is determined bythe sequence specific interaction of specific amino acids of theregulator with the operator DNA. In some systems interactions have beensubject to detailed biochemical analysis as well as high resolutionX-ray crystallography. The best-characterized class of DNA bindingproteins exhibit a common helix-turn-helix motif with some degree ofamino acid sequence homology. It is clear that the critical DNA bindingdomain of the regulator is contained within the helix-turn-helix region.

In eukaryotes it has been shown that control of gene expression is alsoregulated by the interaction of specific protein factors binding to DNAsequences close to the promoter region of genes. A number of factorshave been isolated from yeast and mammalian cells and have been shown tointeract with specific sequence motifs in a sequence-specific mannersimilar to that observed in bacterial systems. Characterization of someof these factors has revealed a new “finger” motif, which may beinvolved in the sequence specific binding of proteins.

Moreover, it has been demonstrated that eukaryotic gene expression canbe controlled through the use of bacterial repressor molecules ineukaryotic cells. In these experiments bacterial operator sequences havebeen inserted close to the promoters of mammalian genes. Cell lines havebeen created which express the bacterial repressor. Control ofexpression of the target eukaryotic genes with operator insertions byrepressor molecules has been demonstrated using transient expressionassays. In these experiments not only repression of gene expression bythe lac repressor has been demonstrated but also induction of geneexpression, that is, relief of repression, using IPTG (isopropylthiogalactoside).

Therefore, detailed knowledge and manipulation of bacterial proteinDNA/interactions can be used to control expression in mammalian cellcultures. Gene switching techniques are described, for example in U.S.Pat. No. 6,010,887, which is incorporated herein by reference. Those ofordinary skill in this art will recognize that other methods of geneswitch regulation may also be employed in the methods and compositionsof the invention.

Although non-genetically modified cells may be used in accordance withthe invention, in one preferred embodiment, the isolated cells aregenetically engineered. The cells can be genetically engineered tosecrete one or more biologically active molecules including, but notlimited to, one or more cytokines, growth factors, and/or angiogenicfactors, or a combination thereof. Examples of such biologically activemolecules are provided in Table 1.

TABLE 1 Target Cells and/ Cytokine Major Source or Major EffectsEpidermal growth Platelets Pleiotropic-cell motility and factor (EGF)proliferation Transforming Macrophages, Pleiotropic-cell motility andgrowth factor epidermal cells proliferation “alpha” (TGFα)Heparin-binding Macrophages Pleiotropic-cell motility and epidermalgrowth proliferation factor Basic fibroblast Macrophages, Angiogenesisand fibroblast growth factor endothelial cells proliferation Acidicfibroblast Macrophages, Angiogenesis and fibroblast growth factorendothelial cells proliferation Keritinocyte Fibroblast Epidermal-cellmotility and growth factor proliferation Transforming Fibrosis andincreased growth factor β tensile strength family (TGFβ) TransformingPlatelets, Epidermal-cell motility, growth factors β1 macrophageschemotaxis of macrophages and and β2 fibroblasts, extracellular matrix(TGFβ1 & TGFβ2) synthesis and remodeling Transforming MacrophagesAntiscarring effects growth factor β3 (TGFβ3) Platelet derivedPlatelets, Fibroblast proliferation and growth factor macrophages,chemoattraction, macrophage (PDGF) epidermal cells chemoartraction andactivation Vascular Epidermal cells, Angiogenesis and increasedendothelial growth macrophages; vascular permeability factor (VEGF)endothelial cells VEGF A Blood vessel Endothelial cell migrationendothelial cells and blood vessel formation VEGF B Blood vesselEndothelial cell migration endothelial cells and blood vessel formationVEGF C Lymphatic vessel Endothelial cell migration and endothelial cellslymphatic vessel formation VEGF D Lymphatic vessel Endothelial cellmigration and endothelial cells lymphatic vessel formation Tumornecrosis Neutrophils Pleiotropic expression of growth factor “alpha”factors (TNFα) Interleukin-1 (IL-1) Neutrophils Pleiotropic expressionof growth factors Insulin-like growth Fibroblasts, Reepithelializationand factor I epidermal cells granulation-tissue formation.Colony-stimulating Multiple cells Macrophage activation and factor 1(CSF-1) granulation tissue formation. Granulocyte Multiple cellsMacrophage activation and Macrophage colony granulation tissue formationstimulating factor (GM-CSF)

Column 1 of Table 1 names suitable biologically active molecules. Column2 displays the major source of the particular biologically activemolecule. Finally, Column 3 shows the target cells and/or major effectof the given biologically active molecule.

Fibroblasts and keratinocytes naturally secrete a vast array of growthfactors and cytokines. A summary of the secretion of various proteins isshown in Table 2:

TABLE 2 Cytokines Keratinocytes Fibroblasts Angiopoietin Yes yes EGF Yesyes Endothelin Yes no FGFs Yes yes FGF-7/KGF Yes yes IFN-α/β no yesIGF-1 no yes IL-1β yes yes IL-6 yes yes IL-8 yes yes IL-18 yes no MCP-1no yes MIP-1α no yes MIP-2 no yes PDGF no yes SLPI yes no TGF-α yes yesTGF-βs yes yes TNF-α yes yes VEGF yes yes

(See Singer, A. and Clark, R. (1999) “Cutaneous Wound Healing” The NewEngland Journal of Medicine 341:738:746.)

Control of the delivery of the secreted biologically active molecule canbe achieved by any method known to those skilled in the art. Forexample, the expression of multiple gene products may be controlled by asingle promoter system. Alternatively, the expression of multiple geneproducts may be controlled by multiple promoter systems, with eachpromoter system regulated either constitutively, by gene switching or bysome combination of both. Further, control over the secretion of aparticular biologically active molecule may be accomplished byup-regulating wild-type gene expression.

A number of well-known transfection methods exist for introducinggenetic material into target cells. These include the use of polycationssuch as DEAE-dextran (see McCutchan, et al., J. Natl. Cancer Inst.41:351-57 (1968) and Kawai et al., Mol. Cell. Biol. 4:1172-74 (1984));calcium phosphate coprecipitation (see Graham et al., Virology 52:456-67(1973)); electroporation (see Neumann et al, EMBO J. 7:841-45 (1982));lipofection (see Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-17(1987)); retrovirus vectors (see Cepko et al., Cell 37:1053-62 (1984));and microinjection (see Capecchi et al., Cell 22:479-88 (1980)).

Moreover, one skilled in the art will recognize that any other methodsuitable for delivering an exogenous biologically active molecule intothe cell types may also be employed in accordance with the invention.

Using any of the above-mentioned transfection methods, control over thesecretion of a variety of biologically active molecules may be achievedby employing any number of cell types secreting the various biologicallyactive molecules.

Additionally, the cell types of the present invention may also be eithermitotically active or mitotically inactive. The term “mitoticallyactive” is used to describe cells that actively undergo mitosis.Conversely, “mitotically inactive” is used to describe cells that do notactively undergo mitosis. Mitotically inactive cells may be growtharrested by any means known in the art. By way of non-limiting example,the cells may be growth arrested by chemical means, such as, forexample, by the administration of mitomycin C. Additionally, the celltypes may be growth arrested by exposure to UV light, X-Ray, or gamma(γ) radiation. In one preferred embodiment, the cell types are growtharrested by exposure to gamma radiation. It is important to note that,e.g., mitotically inactivated, human fibroblast cells terminallydifferentiate and thereby change the pattern of polypeptide expressionand secretion (Francz, Eur. J. Cell. Biol. 60:337-45 (1993)). As afurther example, keratinocyte differentiation usually depends on cultureconditions (including, for example, the composition of culture media,the Ca²⁺-concentration, and whether the cells are cultured at theair-liquid interface), however, keratinocytes may also be induced todifferentiate, e.g., by mitomycin C.

The cell types according to the invention may be autologous, allogeneic,or xenogeneic. Most preferably, the cell types used in the presentinvention are allogeneic. Xenogeneic cells can be isolated for examplefrom transgenic animals expressing molecules of interest.

Stromal cells including, for example, fibroblasts, can be isolated byany method known to those skilled in the art. For example, fibroblastsmay be derived from organs, such as skin, liver, and pancreas. Theseorgans can be obtained by biopsy (where appropriate) or upon autopsy.Specifically, sufficient quantities of fibroblasts can be obtainedrather conveniently from breast reduction, foreskin, or any appropriatecadaver organ.

Fibroblasts can be readily isolated by disaggregating an appropriatesource organ or tissue. By “source organ or tissue” is meant the organor tissue from which the cells are obtained. Disaggregation may bereadily accomplished using techniques known to those skilled in the art.Examples of such techniques include, but are not limited to mechanicaldisaggregation and/or treatment with digestive enzymes and/or chelatingagents that weaken the connections between neighboring cells therebymaking it possible to disperse the tissue into a suspension ofindividual cells without appreciable cell breakage. Specifically,enzymatic dissociation can be accomplished by mincing the tissue andtreating the minced tissue with any of a number of digestive enzymes,either alone or in combination. Suitable enzymes include, but are notlimited to, trypsin, chymotrypsin, collagenase, elastase, hyaluronidase,DNase, pronase, and/or dispase. Mechanical disruption can beaccomplished by a number of methods including, but not limited to, theuse of grinders, blenders, sieves, homogenizers, pressure cells,insonators or trituration. See Freshney, Culture of Animal Cells. AManual of Basic Technique, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch.9, pp. 107-26.

Once the source tissue has been reduced to a suspension of individualcells, the suspension should be fractionated into subpopulations fromwhich the fibroblasts and/or other stromal cells and/or elements can berecovered. Fractionation may be accomplished using standard techniquesfor cells separation including, but not limited to, cloning andselection of specific cells types, selective destruction of unwantedcells (negative selection), separation based upon differential cellagglutinability in the mixed population, freeze-thaw procedures,differential adherence properties of the cells in the mixed population,filtration, conventional and zonal centrifugation, centrifugalelutriation (counter-streaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting. See Freshney, Culture of Animal Cells. A Manual of BasicTechniques, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137-68. Those skilled in the art will recognize that other suitable cellfractionation technique(s) can also be used.

Preferably, the isolation of fibroblasts is accomplished by explantationof skin pieces according to the method of Sly and Grubb. See Sly et al.,Methods Enzymol. 58:444-50 (1979).

Fibroblasts obtained from different source organs or tissues (including,e.g., skin, liver, and pancreas) can be employed in the methods andcompositions of the invention. Moreover, those skilled in the art willrecognize that any such fibroblasts can be genetically engineered tosecrete differing amounts of a biologically active molecule ormolecules.

As used herein, the term “cell preparation” refers to the mixtureresulting from the combination of a preparation of cells that secreteone or more biologically active molecules and a preparation of theextracellular or other matrix materials. For example, a cell preparationaccording to the invention results from the combination of athrombin/cell preparation and a fibrinogen preparation. In someembodiments, the resulting mixture results in polymerization, therebyproducing a cured matrix. Alternatively, the resulting combination mayproduce a highly viscous, non-cured matrix. The resulting cellpreparation can be in the form of a paste. Those skilled in the art willrecognize that as used herein, the term “cell preparation” encompasses aspectrum of mixtures ranging from a viscous, non-cured mixture (i.e. apaste) to a polymerized, cured matrix. Differences in the concentrationof each preparation as well as the culture conditions can influence theviscosity and/or the degree of polymerization of the resulting cellpreparation. For example, in one embodiment of the invention, thecombination of the fibrinogen and the thrombin/cell preparations arespray administered to a wound site to form an irreversible, polymerizedcell matrix. However, those skilled in the art will also recognize thatother combinations may result in a viscous, non-cured cell matrix.

To create the cell preparation according to the invention, the celltypes or genetically engineered cell lines are preferably admixed orcombined with a supporting biological or synthetic extracellular matrixor matrix material (ECM). One skilled in the art will recognize that theterm “ECM” refers to the noncellular material distributed throughout thebody of multicellular organisms. The ECM is comprised of diverseconstituents such as glycoproteins, proteoglycans, complexcarbohydrates, and other molecules. Major functions of the ECM include,but are not limited to, providing structural support, tensile strengthor cushioning; providing substrates and pathways for cell adhesion andcell migration; and regulating cellular differentiation and metabolicfunction. ECM proteins include, for example, collagens, elastin,fibronectin, laminin, proteoglycans, vitronectin, thrombospondin,tenascin (cytoactin), entactin (nidogen), osteonectin (SPARC), anchorinCII, chondronectin, link protein, osteocalcin, bone sialoprotein,osteopontin, epinectin, hyaluronectin, amyloid P component, fibrillin,merosin, s-laminin, undulin, epilligrin, and kalinin. Preferred ECMproteins for use according to this invention include collagen, alginate,agarose, fibrin, fibrin glue, fibrinogen, laminins, fibronectins, HSP,chitosan, heparin and/or other synthetic polymer or polymer scaffolds.

Cell density and the concentration of the extracellular matrix may bevaried for the desired clinical application. For example, certain woundsmay require greater or lesser cell densities and/or differentconsistency preparations. The resulting cell preparations can be in theform of a cell paste or in the form of a cured matrix. Determination ofthe appropriate cell density and concentration of the ECM is within theroutine skill of those in the art. The cell suspension can come from onecell type or can be comprised of a mixture of different cell types. Forexample, the cell mixture may include 50% of keratinocytes and 50% offibroblasts. However, the ratio of keratinocytes to fibroblasts may bevaried, e.g. 1:1, 1:4, 1:9, 1:24 etc. without impairing the formation orthe function of the cell preparation. Alternatively, the cell mixturemay contain only keratinocytes or only fibroblasts (i.e., the ratio ofkeratinocytes to fibroblast may be 1:0 or 0:1). Moreover, the suspensionmay be comprised of more than two different cell types. The percentagesof each cell type in the cell suspension may vary depending on theintended use for the cell preparation. The cell types can also bepre-induced or co-cultured in vitro in order to optimize the healingresponse on the wound. For example, fibroblasts can be pre-incubatedwith TGF-beta (from 0.1 to 30 ng/ml of medium) for 1 to 21 days prior towound application.

The cell preparation of the invention is made from two components. Thefirst, referred to herein as “component #1” is the fibrinogen component.The second, referred to herein as “component #2” is the cells+thrombincomponent. Component #2 can optionally contain a cryoprotectant. Thecell preparation of the invention is formed by the coagulation of plasmaproteins (including fibrinogen) in the presence of thrombin. Thiscoagulation is chiefly due to the formation of a polymerized fibrinnetwork, which imitates the formation of a blood clot. Thrombin convertsfibrinogen to fibrin by enzymatic cleavage. Calcium accelerates theproteolytic activity of thrombin. In fact, the combination of fibrinogenand thrombin results in a “polymerization reaction”. Upon mixing ofthese materials, the cells become entrapped in the resulting cellpreparation, which may be a paste or a cured matrix, depending on theconcentrations of all components, the number of cells, the cultureconditions, etc. Additionally, in any of the cell preparations of theinvention, any or all of the components may also contain additionalproteins or chemicals, which do not affect the formation or function ofthe cell preparation, such as proteins (i.e. Albumin), proteinaseinhibitors (i.e., Aprotinin), polyethylene glycol (PEG), polyvinylalcohol (PVA), and other molecules typically used as stabilizers forcell preparations. In other embodiments of the invention, the firstcomponent of the cell preparation may contain thrombin and the secondcomponent may contain cells plus fibrinogen. Those skilled in the artwill recognize that, in this embodiment, the combination of component #1and component #2 will also result in a cell preparation useful fortissue regeneration. Moreover, in other embodiments, the cellpreparations suitable for tissue regeneration may result from “syntheticpolymerization” rather than from polymerization following theinteraction of fibrinogen and thrombin. In this embodiment, the cellsare mixed with a polymerization agent, either before or afterapplication to the wound site. Once polymerization occurs, a cellpreparation suitable for tissue regeneration may be formed.

While the compositions, cell preparations, kits, and/or methodsdescribed herein refer to the use of a first component containingfibrinogen and a second component containing cells+thrombin, othercomponents may also be used. Examples of such components include thethrombin component #1 and the cells+fibrinogen component #2 as well asthe components leading to synthetic polymerization, which are discussedin detail above. Those skilled in the art will recognize that any of thecompositions, cell preparations, kits, and/or methods described hereinusing the fibrinogen component #1 and the cells+thrombin component #2may also employ any other combination of components which result in acell preparation for tissue regeneration in which the cells becomeentrapped in the resulting paste or matrix, without deviating from thenature of the invention.

Methods of Administration

In one preferred embodiment, the invention involves a combination ofhuman allogeneic fibroblast and keratinocyte cell lines admixed with ECMmaterials to form a viscous cell paste to adhere to a wound. In thisembodiment, the cell lines are preferably not genetically engineered.The cell lines may be mitotically inactivated by any means known tothose skilled in the art. Preferably, the resulting paste is bothbiodegradable and biocompatible. The paste may be applied to the woundas needed, for example, once weekly. Application of the cell pasteaccording to this embodiment facilitates the induction of granulationtissue and the stimulation of wound closure.

As previously described, immortalized fibroblast and keratinocyte celllines would also be preferred embodiments. The preferred immortalizationmethod would be through directly adding the gene for TERT into theprimary human keratinocyte and fibroblast cells such that the TERT geneis constitutively expressed. In addition, a transient immortalizationusing a protein domain transport sequence (TAT, VP22, MTS, etc. attachedto the TERT protein might be more preferable in that the gene is notpermanently inserted into the immortalized cell but is instead added asa fusion protein to the growth medium. In this way, the cell line couldbe continuously expanded, banked, and screened for stable properties(growth rate, factor secretion, etc.), without the continual need forthe revalidation of new primary cell sources. Cell lines immortalized inthis way would preferentially be mitotically inactivated beforeapplication to the wound or tissue repair site as a paste, cellpreparation, biological matrix mixture, or as attached or adsorbed to awound dressing.

The present invention has human clinical and veterinary applications.The cell preparation of the invention can be used to treat humans andnon-human animals, including, a non-human primate, mouse, rat, dog, cat,pig, sheep, cow, or horse. The cell preparation according to theinvention can be used for tissue regeneration such as, e.g., in skinwound treatment or in treatment of peritonitis.

For example, the cell preparation of the invention can be incorporatedinto other pharmaceutical compositions suitable for administration. Suchcompositions can comprise the cell preparation and an additionalacceptable carrier. As used herein, “biologically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial, and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with biologics administration. Suitablecarriers are described in the most recent edition of Remington'sPharmaceutical Sciences, a standard reference text in the field, whichis incorporated herein by reference. Preferred examples of such carriersor diluents include, but are not limited to, water; saline; dextrosesolution; human serum albumin; HBSS and other buffered solutions(including those with and without Ca⁺⁺ and Mg⁺⁺) known to those skilledin the relevant arts; and basal media. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions can be included in a container, pack,kit, or dispenser together with instructions for administration.

The dosage regimen is selected in accordance with a variety of factorsincluding species, age, weight, sex, and medical condition of thepatient; type and severity of the condition to be treated; the route ofadministration; and the particular cells employed. An ordinarily skilledphysician or veterinarian can readily determine and prescribe theeffective amount required to prevent, counter, or arrest the progress ofthe condition.

The cell preparation of the invention can be administered topically tothe wound in need of treatment. The thrombin+cell component (component#2) can be admixed with the fibrinogen component (component #1) before,during, or after application to the wound. In one embodiment, thecomponents are applied to the wound site by simple pipetting or byco-extruding components from a tube or syringe applicator. Eachcomponents may also be applied in conjunction with a sterile gauzedressing. Moreover, those skilled in the art will recognize that theorder of topical administration of the components can be varied (e.g.thrombin+cells component followed by fibrinogen component or fibrinogencomponent followed by thrombin+cells component).

The cell preparation of the invention can also be administered byspraying the components of the preparation onto the wound area. Spraypumps suitable for use in administering the cell preparation of theinvention include those used for other medical applications, includingnasal and throat sprays. Additionally, the cell preparation could beapplied using a spray generated by compressed inert gasses rather thanusing a spray pump. For example, small canisters of medical grade inertgasses such as air and/or nitrogen can be used. The pressure of the gascan be used to propel fluids through a small orifice, thereby generatinga fine mist spray. The pressure can act directly on the fluid or apressure drop can pull fluid out into an air stream, which eventuallywould become a spray.

In another embodiment, the cell preparation of the invention may bespray administered as a two component product that is applied to achronic ulcer in a sequential, two-step process. In this embodiment, thefirst component is a suspension of fibrinogen in HESS (without Ca⁺⁺ andMg⁺⁺) and the second component is a mixture of cells (includingfibroblasts and keratinocytes), thrombin and cryoprotectants in HBSS(with Ca⁺⁺ and Mg⁺⁺). In this embodiment, the two components of the cellpreparation are mixed together on the target wound area. Both componentsare applied to the chronic ulcer or wound using a spray applicator, suchas a spray pump. For example, a spray pump may be used to deliverprecise doses of both components during treatment of an ulcer or wound.The actual design of the spray pump used may vary depending on themanufacturer. Examples of suitable spray pumps include for example nasaland throat spray. The spray pump is manufactured of a material that canbe sterilized using conventional techniques to avoid contamination ofeither component of the cell preparation.

Sprayed doses can range from about 50 μl to about 150 μl per spray,preferably from about 100 μl to about 150 μl per spray, most preferablyabout 130 μl per spray. To allow for concentrated application and formore precise delivery of the product, the spray pump should have a sprayactuator mechanism that produces a narrow spray diameter rather than alarge diameter spray. The spray actuator mechanism is the “arm” thatorients and generates the spray via the orifice size. The area of thechronic ulcer or wound surface area covered by each spray dependsdirectly on the distance of the actuator from the target. For example,the closer the spray actuator mechanism is to the target area, thesmaller the surface area covered per spray. Likewise, the further awayfrom the target area the spray actuator mechanism is, the larger thesurface area covered per spray.

Preferably, the surface area covered ranges from about 11 cm² to about14 cm². In one preferred embodiment, the distance between the spraynozzle and the target is approximately 6 cm. At this distance, using anarrow diameter spray actuator mechanism, one spray will cover a woundsurface area of approximately 12.6 cm².

The number of cells landing on the target area of the patient (i.e., thenumber of cells per cm² of patient) will vary depending on theconcentration of each of the components and the ratios of keratinocytesto fibroblasts used in component #2. Those skilled in the art willrecognize that the concentration of cell in the second component of thecell preparation can be varied from about 1×10³ cells/μl to about 50×10³cells/μl. For example, in some embodiments, the number of cells/μl ofthe cell preparation component #2 can range from about 5×10³ cells/μl toabout 20×10³ cells/μl. Thus, if two sprays of approximately 130 μl/sprayare administered to a patient, approximately about 1.3×10⁶ to about5.2×10⁶ cells are administered to the patient.

Typically, the first component (fibrinogen) is sprayed onto the targetfollowed by spraying the second component (thrombin+cells). The order ofspraying of the components can be reversed. However, it is preferable tofirst apply the fibrinogen component and then subsequently apply thethrombin+cells component, which may optionally contain a cryoprotectant.Once the cells+thrombin component is sprayed onto the fibrinogencomponent, the two components will begin to gel, cure or polymerizealmost immediately, allowing an equal distribution of cells on thetarget area. When the cells+thrombin component is applied first followedby the fibrinogen component, the resulting lag time allows the cells tomigrate on the wound site due to the effects of gravity, which mightcause the cells+thrombin component to “run” or “drip” after application,depending on the volume applied. This, in turn, could potentially leadto an unequal distribution of cells upon application of the fibrinogencomponent. Since the subsequent application of the fibrinogen componentleads to polymerization, this could result in the formation of an unevencell preparation.

In another embodiment, the cell preparation components can beadministered as a spray that is mixed in the air prior to reaching thetarget area. In such an embodiment, two separate components could besprayed at the same time using one or two spray actuator mechanisms. Thespray mists of each component would then combine in the air, therebyinitiating polymerization before the cell preparation reaches the targetarea (i.e., the wound site). This embodiment would make the cellpreparation of the invention easier to apply, as it requires a singlespray to apply the cells and to initiate fibrin polymerization.

Cryopreservation

In some embodiments, component #2 cells may be cryopreserved prior touse in the cell preparation of the invention. In such embodiments,component #2 contains cells+thrombin+a cryoprotectant. Those skilled inthe art will recognize that, the terms “cryoprotectant” and“cryopreservant” are used interchangeably herein and cover agents usedto achieve cryopreservation. Suitable cryoprotectants include, e.g.,glycerol, ethylene glycol, and dimethyl sulfoxide (“DMSO”). In variousembodiments, the cryoprotectant includes, but is not limited to a 10%glycerol solution, a 15% glycerol solution, and a 15% glycerol and 5%human serum albumin (HSA) solution. Those skilled in the art willrecognize that any other cryoprotectants and cryoprotectantconcentrations known in the art may also be used.

The cryoprotectants used in the instant invention can be included in thebuffers containing component #2 of the cell preparation. In thisprotocol, no extra proteins need to be added. Likewise, the cells arenot frozen in biological media. By choosing a cryoprotectant with low orno toxicity, there is no need to wash away or otherwise remove thecryoprotectant from the cells prior to use. This allows directapplication of the cell preparation after thawing.

Cryopreservation facilitates shipping and long-term storage of thecomponents of the cell preparation of the invention. Cryopreserved cells(in component #2) are stored at a temperature ranging from about −70° C.to about −196° C. (if liquid nitrogen is used). For example, thecryopreserved cells may be stored in a −80° C. freezer or in the vaporphase of liquid nitrogen at −160° C.

In one preferred cryopreservation protocol, a vial containing thecell+thrombin+cryoprotectant mixture (component #2) is closed with ascrew-on closure in a sterile manner. The closed vial is then packaged(hermetically sealed) inside a pouch fabricated of a material that canresist temperatures ranging from −70° C. to −196° C., which is thetemperature found in the liquid nitrogen vapor phase. Other storagetemperatures between −120° C. and −160° C. can be found in the liquidnitrogen vapor phase. Vials or pouches containing vials are then placedinside a controlled rate freezer employed for the freezing of biologicalcells and tissues in a special rack designed to be inserted inside thecontrolled rate freezer. Typically, the vials or pouches are storedupright and aligned in several rows, such that there is space betweeneach row. The rows are aligned parallel to the flow of the liquidnitrogen vapor that passes through the chamber to cool the samples. Theuse of such pouches helps to prevent contamination of the componentsduring freezing and thawing of component #2.

After loading the samples into the chamber, a freeze cycle is initiated,as follows:

-   -   1. Beginning at room temperature or 20° C., the chamber is        cooled at −2° C. per minute until 4° C.    -   2. The chamber is then held at 4° C. for 52 minutes to stabilize        all samples at 4° C.    -   3. The chamber is then cooled at −1° C. per minute until −5° C.    -   4. The chamber is then cooled at −3° C. per minute until −12° C.    -   5. The chamber is then cooled at −7.5° C. per minute until −20°        C.    -   6. The chamber is then held at −20° C. for 15 minutes.    -   7. The chamber is then cooled at −1° C. per minute until −80° C.    -   8. The chamber is then held at −80° C. for 60 minutes.

The pouches are then removed from the freezer and stored at either −160°C. or −80° C. until the time of use.

Those skilled in the art will recognize that the exact method used inthe cooling cycle may be changed or improved upon as necessary. Ingeneral, it is well known that cells or tissues should be cooled at arate ranging from about −0.2° C./minute to about −5° C./minute.Moreover, cooling rate ranges of about −0.5° C./minute to about −2°C./minute are optimal for most cases.

The cooling rate is especially critical as the temperature is lowered tothe freezing point of the cryoprotectant solution used. In the preferredembodiment described herein, cooling is accelerated around the freezingpoint of the solution. This global, rapid drop in chamber temperature isintended to induce the phase change by generating a thermal instabilitythat would induce ice seeding. It is desirable to control the seeding ofthe phase transition process in order to initiate the phase transitionof all samples at the same time. Seeding of the phase transition processmay be controlled by several methods, including: creation of a pointthermal instability by either (1) touching a sample with a metal probechilled in liquid nitrogen or (2) ejecting a cooled liquid (e.g. liquidnitrogen) at the sample. (See U.S. Pat. Nos. 6,347,525; 6,167,710; and5,981,617, each of which are incorporated herein by reference). Thoseskilled in the art will recognize that other means of controlling thephase transition can also be used. For example, any available techniquethat creates an instability in the supercooled solution such asmechanical stimulation (e.g. a pulsed vibration) can be used.

In some embodiments, the cell preparation of the invention can bedelivered as a kit contained in a single package made from an aluminumfoil laminate pouch that resists low temperatures. Inside this outerpouch are two smaller pouches, the first with a vial containingcomponent #1 (fibrinogen) and the second with a vial containingcomponent #2 (cells+thrombin+cryoprotectant). Alternatively, vials forcomponents #1 and #2 can be packaged together in a single pouch or case.These inner pouches are also made from a temperature resistant material.Examples of suitable materials for these pouches include, but are notlimited to, an aluminum foil laminate pouch, coated paper, LDPE,Surlyn®, and a Kapton™ laminate pouch (Steripack, Ireland). Thoseskilled in the art will recognize that any other suitable material mayalso be used to make these pouches. The material used to manufacture thepouch should have a low seal initiation temperature, high barrierperformance, and good chemical resistance. Moreover, it should besuitable for irradiation sterilization to avoid contamination of thecell preparation components. An example of the composition and typicalproperties of a suitable pouch are provided in Tables 3 and 4.

TABLE 3 STRUCTURE Thickness (micron) Weight (gm/m²) Paper — 50Polyethylene 13 12 Aluminum Foil  9 25 Surlyn ® — 23

TABLE 4 Value PROPERTY UNITS (typical) Tensile Strength (MD) KN/m² >50.0Tensile Strength (CD) KN/m² >20.0 Elongation (MD) % >1.0 Elongation (CD)% >2.0 Lamination Strength T: 140° C., P: 4 bar, t: 0.3 sec >5.0 WaterVapor g/m² · d @ 38° C., 90% R.H. <0.5 Transmission Rate Oxygen (O₂)cc/m² · d · atm@ 23° C., 50% R.H. <2.0 Transfer Rate

The sealing parameters for a suitable pouch will depend on theparticular sealing equipment used and its compatibility with the sealantlayer of the secondary substrate. However, typical parameters forsealing can include: temperature=100-140° C.; dwell time=0.30-0.75 sec;and pressure=60-80 psi.

In another embodiment, the vials may be sealed in a rigid, transparentcontainer, fabricated by using a polymer resistant to temperatures below−70° C. to as low as −196° C. A variety of different types of vials maybe used for freezing components #1 and #2. In one preferred embodiment,a novel vial (5) such as that shown in FIG. 6 a and FIG. 6 b isemployed. This vial is used to freeze down the components of the cellpreparation of the invention. Upon thawing, the screw-on cap (4) closingthe vial can be replaced with a spray pump applicator. In oneembodiment, bottle may be made of polypropylene, which is resistant tothe low temperatures employed in the cryopreservation protocol. The wallthickness of this vial (1) should be approximately 0.8 mm to facilitateheat/cold transfer across the wall, which is important for both thefreezing and thawing processes. Additionally, the vial can be designedto stand upright after a spray pump has been screwed on, and the bottomof the vial (2) is conical (3) to facilitate emptying of the contents.

One spray pump designed to be used with this type of vial ismanufactured by the company Valois. Each spray delivers a 130 μl volumeof product. The spray actuator, which is the “arm” that orients andgenerates the spray via the orifice size, can be modified so that the“arm” can be oriented in directions other than the horizontal position,to aid in topical administration by allowing spray application onto ahorizontal surface without tipping the bottle. In another embodiment,other spray pump designs can be employed which allow the spray tofunction when the bottle is inverted.

The cryopreserved components of the cell preparation of the inventioncan be shipped, stored, and/or used as follows. The components may beshipped as a kit frozen on dry ice at a temperature of about −70° C. toabout −80° C. The pump may be shipped at room temperature. Upon arrival,the kit should be stored in a −80° C. freezer or at −160° C. until use.To use, the outer pouch of the kit is opened and the two smaller pouchescontaining component #1 (fibrinogen) and #2(cells+thrombin+cryoprotectant) are removed and thawed in a water baththat is warmed to a maximum of 37° C. Those skilled in the art willrecognize that the pouches serve to prevent water in the water bath fromcontaminating the components of the cell preparation. Once the contentsof the vials are thawed, the pouches can be removed from the water bath,disinfected (if desired), and opened. The screw-on top from each vial isthen removed and replaced with a screw-on spray applicator, and the cellpreparation components are then ready for patient application.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1 Isolation of Keratinocytes and Fibroblasts

Keratinocytes and fibroblasts may be isolated after splitting theepidermis from the dermis using an enzyme such as dispase orthermolysin. The isolated epidermis can be incubated with trypsin toobtain a single cell suspension of keratinocytes, which can then beplated onto culture dishes and amplified to create a bank of primarykeratinocytes. The isolated dermis can be incubated with a dissociationenzyme such as collagenase to obtain fibroblast single cell suspensionsready to be plated and amplified or minced and dispatched onto a cultureplate, and cultured until fibroblasts have migrated from the tissue.These cells can then be collected after trypsin treatment and furtheramplified to establish a fibroblast cell bank. Primary humankeratinocytes and fibroblasts isolated in this manner can be used forthe preparation of cell and fibrin admixtures.

The isolation of fibroblasts may also be carried out as follows: freshtissue samples are thoroughly washed and minced in Hank's balanced saltsolution (HESS) in order to remove serum. The minced tissue is incubatedfrom 1 to 12 hours in a freshly prepared solution of a dissociatingenzyme such as trypsin. After such incubation, the dissociated cells aresuspended, pelleted by centrifugation and plated onto culture dishes.All fibroblasts will attach before other cells, therefore, appropriatestromal cells can be selectively isolated and grown. The isolatedfibroblasts can then be grown to confluency, and serially cultured orstored frozen in liquid nitrogen (see, Naughton et al., 1987, J. Med.18(3&4):219-250). Fibroblasts or subpopulations of fibroblasts such asdermal papilla cells or myofibroblasts can be isolated from explantoutgrowth culture. Once isolated, the stromal cells are ready foradmixture with an extracellular matrix (e.g. fibrin) paste.

Example 2 Composition of the Components of the Cell Preparation of theInvention

Component #1

This component can be made by performing a four-fold dilution of theSealer Protein Solution in the Tisseel VII Fibrin Sealant (Baxter).After dilution, the concentration of the components in the SealerProtein Solution was as follows:

Fibrinogen: 18.75 mg/ml-28.75 mg/ml Fibrinolysis Inhibitor (Aprotinin):750 KIU/ml Polysorbate: 0.05 mg/ml-0.1 mg/ml  Sodium Chloride: 0.5mg/ml-1.0 mg/ml Trisodium Citrate: 1.0 mg/ml-2.0 mg/ml Glycine: 3.75mg/ml-8.75 mg/ml

The Sealer Protein Solution was diluted in Hank's Buffered SalineSolution (HBSS) without Ca²⁺ or Mg²⁺. The presence of these two ionsinduced the formation of precipitates during the freezing and thawingprocess.

Those skilled in the art will recognize that the Tisseel Sealant can besupplemented with other commercially available fibrinogen and aprotininpreparations to achieve similar results. Moreover, fibrinogen may alsobe diluted to other concentrations, depending on the mode ofadministration, to enhance the polymerization characteristics.

Component #2

This cryoprotected component can be made by mixing the following:

Components Final Concentration Thrombin (Baxter Tisseel): 10% by volumeGlycerol 15% by volume Human Serum Albumin  5% by volume HBSS (with Ca²⁺and Mg²⁺) Used to resuspend these components

Following dilution, the thrombin solution obtained from Baxtercontributes the following to the cryoprotected solution:

Thrombin: 50 IU/ml Total protein: 4.5-5.5 mg/ml Sodium Chloride: 0.8-1.2mg/ml Glycine: 0.24-0.36 mg/ml CaCl₂: 4 μmol

HBSS with Ca²⁺ and Mg²⁺ was the chosen diluent.

The desired cell mixture (ratio) and concentration was resuspended inthe cryoprotected solution to obtain the component #2. Variouskeratinocyte:fibroblast ratios have been considered, including 1:1, 1:3,1:4, 1:9, to as high as 1:50. Moreover, various final cellconcentrations in component #2 considered have been 1 million, 2.5million, 5 million, 10 million, 20 million, and 50 million cells/ml(final concentration).

Example 3 Testing the Effective Dose of Mitomycin C

Previous work has shown the efficient concentrations of mitomycin C(MMC) for growth-arrest of mouse 3T3 fibroblasts to be 2 μg/ml(Rheinwald and Green, Cell 6:331-43 (1975)) and for growth arrest ofhuman dermal fibroblasts to be 8 μg/ml (Limat et al., J. Invest.Dermatol. 92:758-62 (1989)).

The rat fibroblast cell line (CRL 1213), the FGF1-transfected ratfibroblast cell line (1175/CRL 1213), the human telomerase immortalizedfibroblast line (MDX12), and the primary human fibroblasts (EDX1) wereeach growth arrested using the following method. Fibroblasts were grownin DMEM+10% FCS, 25 mM Hepes, 1 mM pyruvate, 2 mM L-Gln, 100 U/mlpenicillin, 100 μg/ml streptomycin in T75 flasks. At confluency, thecells were detached and plated at a density of 10⁵ cells/cm², furtherincubated for 48 h, then treated with mitomycin C (MMC) at 0, 2, 4, 8,12 μg/ml for 5 h. The cells were then rinsed with PBS and detached with0.05% trypsin/0.02% EDTA. The remaining cells were plated at densitiesof 100 to 5000 cells/cm² in T25-flasks respectively (10 flasks for eachdensity). These cells were incubated at 37° C. with 2 media changes perweek.

Efficiency of cell growth arrest was measured by weekly counting ofcells (using a hemacytometer) cultured in the flasks plated at a densityof 5000 cells/cm² and by inspection of appearing colonies in the flasksplated at a density of 100 cells/cm². Changes in cell morphology werealso examined. A concentration of 8 μg/ml MMC was sufficient to growtharrest the human primary fibroblasts (EDX1) and the hTERT immortalizedhuman fibroblast line (MDX12) agreeing with previous data (Limat et al.,J. Invest. Dermatol. 92:758-62 (1989)). The rat fibroblast line (CRL1213) showed a toxicity to MMC-concentrations above 4 μg/ml, while 2μg/ml MMC proved to be optimal to growth arrest these cells. The ratfibroblast cell line (1175/CRL 1213) transfected with the FGF1-gene, wasgrowth arrested at a concentration of 1 μg/ml MMC. Concentrations below1 μg/ml were not efficacious and higher concentrations of MMC wereprogressively toxic. Mitomycin C-treated fibroblasts (with theappropriate mitomycin-C dose), recovered from cryogenic storage bythawing, displayed a cell recovery of at least 50% (in agreement withLimat et al., In Vitro Cell Dev. Biol. 26:709-12 (1990)).

Example 4 Testing Effective Dose of Gamma Irradiation on Fibroblasts andKeratinocytes

To measure the effect of gamma (γ) irradiation on the mitotic activityof treated fibroblasts, a BrdU cell proliferation assay was employedwhich functions in a 96 well format (Oncogene Research Products).Non-immortalized, human primary fibroblast cells were treated with γirradiation at various doses, including 0, 60, 70, and 80 Grays. Cellswere then plated at a density of 5,000 cells per well in a 96 well dish.Irradiated and non-irradiated controls were maintained in culture forperiods lasting 15 and 30 days. At each of these time points, themitotic activity was measured using a BrdU incorporation assay in whichBrdU incorporation in the DNA is assayed immunochemically and measuredvia its absorbance at 450 nm.

In FIG. 1, non-irradiated cells treated with BrdU served as a positivecontrol for each experiment. Non-irradiated cells that were not treatedwith BrdU served as the background absorbance, which was subtracted fromthe absorbance at 450 nm for each sample (0, 60, 70, and 80 Grays) andthe final data was normalized by setting the positive control to 100%relative BrdU incorporation. In FIG. 1, it is clear that treatment withγ irradiation at levels of 60 Grays and above induces primaryfibroblasts into a post mitotic state. A total of 3 samples wasconsidered for each condition. Data is shown as the average±SEM.

In FIG. 2, viability of cells following gamma (γ) irradiation treatmentwas assessed by determining the adhesion of cells to normal cell culturesurfaces. Cells were plated in 6 well culture dishes at a concentrationof 9,500 cells/cm² or 95,000 cells per well. Four hours after platingcells, media was replaced with a fresh media containing 50 μg/ml ofNeutral Red and cells were incubated for 2 hours at 37° C. and 5% CO₂.Wells were then rinsed twice with NaCl 0.9% and dried overnight. Thefollowing day, the dye was dissolved using a mix of 1:1 acidic acid (2%)and ethanol (95%). Each well was incubated in 1 ml of this solution atroom temperature for 15 minutes after which 200 ul of the mix wasmeasured for its dye content at 540 nm. No significant difference wasobserved between the adherence of treated versus non-treated cellsindicating that gamma (γ) irradiation does not effect cell viability andthat cells may attached to a culture surface following gamma (γ)irradiation. Each data point is the average measurement of threeseparate 6 wells. Data is displayed as the average±SD.

Gamma (γ) irradiation at 80 Gy was also evaluated for its ability toinduce differentiation of primary human keratinocytes into a postmitotic state. Irradiated keratinocytes were plated on a layer of growtharrested fibroblasts feeder cells (gamma (γ) irradiated at 70 Gy).Feeder cells were plated at a density of 5,000 cells/cm² andkeratinocytes 12,500 cells/cm². Keratinocyte growth and phenotype werefollowed for 3 weeks thereafter by observation using an invertedmicroscope. During this period, keratinocytes were observed to adopt adifferentiated phenotype, with cells increasing their size and area ofattachment. Keratinocytes cultured in this manner were not able todivide and cover the culture surface, but instead remained eitherisolated or in small clusters, indicating that irradiation had inducedcells into a post mitotic state.

Example 5 Testing Cell Densities with Fibrin Paste: Secretion of GrowthFactors and Cytokines by Mixtures of Keratinocytes and Fibroblasts in aFibrin Matrix

Human primary keratinocytes and fibroblasts were growth arrested bygamma (γ) irradiation at 80Gy prior to formulation. Fresh preparationsof human primary keratinocytes and fibroblasts were mixed at a ratio of1:9 at final concentrations including 2.5, 5, 10, 20 and 40 millioncells/ml in a suspension containing 10% thrombin (Tisseel, Baxter)+15%glycerol+5% Human serum albumin. In a second vial, a 25% fibrinogen(Tisseel, Baxter) solution was prepared. A cell and fibrin “paste” wasprepared in individual wells of a 24 well plate by spraying together 1spray (130 μl) of the cell+thrombin suspension with 1 spray (130 μl) ofthe fibrinogen suspension. Secretion of various growth factors andcytokines by these cells into media was measured during day 2 followingpreparation of the cells in the fibrin matrix. Data shown in Table 5represents an average of 5 individual data points (samples) for eachcondition presented.

TABLE 5 Cell GM-CSF (pg) VEGF (pg) KGF (pg) HGF (pg) IL-1 beta (pg)Concentration Average SEM Average SEM Average SEM Average SEM AverageSEM 2.5 million/ml  9 5 1002 80 90 6 1869 144 29 4  5 million/ml 42 143528 385 207 9 3533 417 58 6 10 million/ml 373 55 11739 822 332 15 5726213 119 8 20 million/ml 963 110 12637 1064 214 34 4956 563 276 51 40million/ml 602 82 4432 733 98 11 2140 197 927 45

Table 5 illustrates the variety of different growth factors are activelysecreted from keratinocytes and fibroblasts contained in a fibrinmatrix. It is also known in the art that bFGF produced by these cellscan be found in the fibrin matrix. The absolute levels of growth factorsproduced were observed to be dependant on the particular nature of thegrowth factor in question. Because biological potency and half-life ismolecule dependant, actual pg levels of independent growth factors isnot the primary interest. Rather, it is believed that the biologicalaction of the cocktail of molecules secreted from these cells togetheroffers a unique way of targeting many biological pathwayssimultaneously. It is worth noting that physiological quantities ofgrowth factors and cytokines are being produced.

In Table 5, the secretion of the 5 different molecules appears to bedose-dependant according to the cell concentration employed. This holdstrue as cell concentration increases from 2.5 to 10 million cells/ml.For most factors, except KGF and HGF, even higher secretion levels arewitnessed at 20 million cells/ml. However, once the cell numberincreases to 40 million cell/ml, a drop in protein production isobserved for all molecules except IL-1 beta. This suggests that anoptimum cell concentration likely exists and that, as shown, each cellconcentration will lead to the production of a different protein profileas seen in the Table 6 below. Table 6 was generated by normalizing thesecretion of VEGF, KGF, HGF and IL-1 beta to that of GM-CSF. Thisillustrates that, for different cell concentrations, there exist adifferent protein profile, with molecules being produced at differentratios depending on the cell concentration under consideration.

TABLE 6 GM-CSF VEGF KGF HGF IL-1 beta 2.5 million/ml  1 111.3 10 207.63.2  5 million/ml 1 84 4.9 84.1 1.4 10 million/ml 1 31.5 0.9 15.3 0.3 20million/ml 1 13.1 0.2 5.1 0.3 40 million/ml 1 7.4 0.2 3.6 1.5

Example 6 Allox Phase I Clinical Trial C2001

Product Description

The allogeneic cell-based treatment (named Allox) under investigation isa two component product which can be applied topically to chronic ulcersusing a spray applicator. Upon spraying the two components on the woundsite, a fibrin matrix is created that traps the applied cells at theregion of the ulcer, permitting local release of trophic factors bythese cells.

The contents of the 2 components were:

-   -   Component #1 A liquid suspension of fibrinogen; (1 spray=50 μl)    -   Component #2 A liquid suspension of keratinocytes and        fibroblasts (Ratio 1:1, 15×10⁶ cells/ml) mixed with thrombin; (1        spray=50 μl)        Objective

The objective of this study was to assess the effects Allox on the woundhealing of chronic ulcers, to assess its safety and tolerability in apatient population, and to determine the effect of the product on theincidence of complete wound closure of chronic leg ulcers in patientswith venous or arteriovenous insufficiency.

Methodology

Patients over 18 years of age with at least one venous or combinedarterio-venous ulcer between the ankle and the knee who met theprotocol's eligibility criteria were recruited to receive Allox in theC2001 study. At study day (SD)—14, patients were found eligible andrecruited to receive the Allox treatment. At SD1, patients received thefirst application of the study treatment, which was repeated on a weeklybasis up to 8 weeks, or until ulcer closure, whichever came first. Afollow-up visit occurred 4 weeks following the last application of thetreatment.

Patient Number

A total of 13 patients were initially enrolled, one of whom wasdetermined to be not eligible for the protocol at a later time.

Diagnosis and Main Criteria for Inclusion

Patients over 18 years of age with one or more venous or combinedarterio-venous ulcers between the ankle and the knee whose etiology wasconfirmed by Ankle Brachial Pressure Index, by Ankle and/or Great Toepressure, and by Duplex Ultrasound were eligible for this study. AtSD-14 patients had to have an ulcer longer than 1 month duration with asurface area greater than 1 cm². Patients were required to be capable ofcommunicating and cooperating with the Investigator and other staff andhad to provide informed written consent. Female patients must have beenpost-menopausal or surgically sterilized.

Test Treatment and Mode of Administration

Application by sequential spraying of the solutions provided in the twobottle kit. A total 0.4 ml of product was applied alternatively: 2sprays of component #1 (fibrinogen) followed by 2 sprays of component #2(cells+thrombin). This procedure was repeated twice. Such an applicationcovers 12 cm² of ulcer area, and each cm² of treated wound received atotal of 0.25×10⁶ cells.

Duration of Treatment

At SD1, patients received the first application, which was repeated on aweekly basis up to 8 weeks, or until ulcer closure, whichever camefirst. A follow-up visit would occur 4 weeks following the lastapplication. The study duration period was 14 weeks, consisting of a2-week run-in period, an 8 week treatment and a follow up period of 4weeks.

Criteria for Evaluation

Efficacy was assessed by noting complete closure of the ulcer; ulcersurface area at W8 and W12 compared to SD1; edge effect; and ulcersymptoms.

Safety was monitored by following the frequency and severity of adverseevents until week 12. Standard laboratory tests, physical examinationsand vital sign measurements were also recorded.

Statistical Methods

Given the small number of patients treated, no statistical analysis wasperformed. Rather, results are presented herein in descriptive form.

Results

TABLE 7 ULCER SURFACE REDUCTION IN INDIVIDUALS PATIENTS TREATED WITHALLOX Ulcer surface Ulcer surface Ulcer surface area (cm²) at % of area% of area area (cm²) at area (cm²) at 4 Weeks reduction reductionPatient ID Center SD1 SD57 Follow Up SD1-SD57 SD1-Follow Up 200111 150.4 52.0 55.2 −3 −10 200113 1 14.0 15.4 12.5 −10 10 200114 1 44.0 50.962.8 −16 −43 200124 2 21.5 20.4 19.9 5 7 200125 2 11.8 10.2 9.8 14 16200126 2 7.7 16.2 16.2 −110 −110 200127 2 10.6 6.5 5.8 38 45 200129 264.5 72.6 73.5 −13 −14 200141 4 10.3 5.5 0.5 47 95 200142 4 20.0 14.010.3 30 48 200143 4 0.8 0.0 0.0 100 100 200144 4 1.8 0.1 0.0 92 100

A total closure was observed for one ulcer at SD57 and for two ulcers atthe week 4 follow-up. For 5 patients, an improvement defined as morethan 30% decrease in surface area was noted. Treatment failure, whichwas defined as either no reduction in ulcer size or an increase of ulcersize during the study period, was observed in 4 patients treated withAllox.

Clinical Study Conclusions

Weekly Allox treatments over an 8 week period were determined to be safeand non-toxic to patients with chronic leg ulcers. The two ulcerinfections that were potentially treatment related resolved before thestudy end. Mild ulcer infections are relatively common place if correcthygiene is not respected.

Five patients showed greater than 30% reduction of area at the end ofthe follow up period, with 2 patients displaying complete ulcer closure.From this small study, a tendency towards response was observed in“younger” ulcers (<6 months).

Example 7 Testing Optimal Dilutions of Tisseel VII (Fibrin Glue) andStandard Human Plasma

Fibrin is one potential biological polymer that can be employed forsuspending and trapping cell mixtures for therapeutic purposes.Polymerized fibrin is created by mixing fibrinogen and thrombin togetherat appropriate concentrations. In the matrix shown in FIG. 3, variousdilutions of the fibrinogen (Tisseel, Baxter) and thrombin (Tisseel,Baxter) were tested for their effect on the polymerization process andgeneration of the end product fibrin. Dilutions considered ranged from ¼to 1/80 of the original fibrinogen and thrombin components supplied inthe TissuCol kit. In the original Tisseel kit, fibrinogen had aconcentration of 75 to 115 mg/ml, while thrombin had a concentration of500 IU/ml. For this study, the fibrinogen was diluted in HBSS withoutCa²⁺ and Mg²⁺ and the thrombin was diluted in HBSS with Ca²⁺ and Mg²⁺.

Fibrin characteristics considered included polymerization time(seconds), consistency, and mechanical strength. The fibrin polymer wasgenerated using a spray technique by which one spray of fibrin wascombined with one spray of thrombin in a single well of a 24 wellculture plate. Conditions were repeated in triplicate. The spray volumeemployed was 130 μl per spray.

All dilutions considered permitted the formation of a fibrin polymer,though the properties of the fibrin polymer varied widely depending onthe dilutions employed. The consistency and the mechanical strength ofthe fibrin were rated using the scale (−, +, ++, +++) in witch − wasconsidered poor and +++ was considered excellent. Ratings of or +++ wereconsidered to be acceptable for potential use to suspend cells fortherapeutic applications. The conditions for the maximal dilution offibrinogen was 1/20 fibrinogen and ⅛ thrombin, while the conditions forthe maximal dilution of thrombin was ¼ fibrinogen and 1/40 thrombin.

Additionally, normal plasma can also be used as a matrix material forthe production of a biological glue to trap cells at the applicationsite of a wound. Mixtures made by pipetting normal undiluted humanplasma together with thrombin at a dilution of 1/50 permitted theformation of a fibrin clot or polymer. This fibrin polymer showedhandling characteristics similar to the commercially available fibringlues, suggesting that it can serve as a substitute to Baxter's TisseelVH or Tissucol and Haemacure's APR concentrated fibrin-based products.

Example 8 Comparison of Growth Factor Release from Sprayed VersusNon-Sprayed Cells

FIG. 4 shows the secretion of growth factors from cells entrapped in afibrin matrix, according to the methods of the instant invention. VEGFand GM-CSF secretion was assessed from medium conditioned for 24 hoursduring the second day following production of the fibrin cell matrix.bFGF secretion was measured in extracts obtained from the cell matrix 48hours after production. To growth arrest cells, both primary humanfibroblasts and keratinocytes were treated with 8 ug/ml mitomycin duringfive hours. Cells were rinsed with HBSS without Ca²⁺ and Mg²⁺ prior totrypsinization.

Cells were applied using a spray pump delivering 50 μl per spray.

Keratinocyte: Fibroblast Ratio=1:4

2 sprays (1.5×10⁶ cells): 100 μl (15 million cells/ml) (cells+thrombin)component+100 μl fibrinogen component.

4 sprays (3×10⁶ cells): 200 μl (15 million cells/ml) (cells+thrombin)component+200 μl fibrinogen component.

Non-sprayed (1.5×10⁶ cells): 100 μl (15 million cells/ml)(cells+thrombin) component+100 μl fibrinogen component.

Keratinocyte: Fibroblast Ratio=1:1

Non-sprayed (pipetted) (2×10⁶ cells): 250 μl (8 million cells/ml)(cells+thrombin) component+250 μl fibrinogen component.

FIG. 4 compares the quantity of secreted growth factors produced by celland fibrin preparations following spray of different cell doses intoindividual wells of a 24 well culture dish. The figure also shows growthfactor secretion quantities when cell and fibrin preparations are madeby simple pipetting (non-sprayed). A comparison of sprayed versusnon-sprayed preparations indicates that there is a decrease in measuredVEGF secretion, while GM-CSF and bFGF secretion levels are comparable.Additionally, an increase from 2 to 4 sprays led to higher secretionlevels of growth factors by the cells, which highlights the possibilityof dosing growth factors by altering the number of cells. SecretedGM-CSF and VEGF were dosed in the culture media, while bFGF was dosed inthe fibrin matrix. Data is presented as the average+SEM (n=4, spray;n=3, non-sprayed).

Example 9 Comparison of Growth Factor Production by DifferentKeratinocyte:Fibroblast Cell Ratios

In FIG. 5 a and FIG. 5 b, a comparison is made of growth factorsreleased from cells entrapped in a fibrin matrix. The fibrin cell matrixis formed by spraying either one or two sprays of component #2(cells+thrombin) with one or two sprays of component #1 (fibrinogen). Aconcentration of 15×10⁶ cells/ml in component #2 was employed and thespray pump used delivered a volume of 70 μl per spray. To growth arrestcells, both primary human fibroblasts and keratinocytes were treatedwith 8 ug/ml mitomycin during 5 hours. Cells were rinsed with HBSSwithout Ca²⁺ and Mg²⁺ prior to trypsinization.

In FIG. 5 a, one spray (1.05×10⁶ cells)=1 spray of cells+thrombin (70μl) and 1 spray of fibrinogen (70 μl). In FIG. 7 b, two sprays (2.1×10⁶cells)=2 spray of cells+thrombin (140 μl) and 2 sprays of fibrinogen(140 μl).

FIGS. 5 a and 5 b demonstrate that mixing keratinocytes and fibroblastsat different ratios, while maintaining a constant total number of cells,gives rise to variable growth factor secretion characteristics. Askeratinocytes are added to the fibroblasts, an increase in GM-CSFsecretion is observed. For one spray, at keratinocytes:fibroblastsratios of 1:24 to 1:8 a plateau is reached for GM-CSF production.Further addition of keratinocytes to the second component of the cellpreparation of the invention does not appear to provide an advantage interms of GM-CSF secretion.

Considering two spray preparations, at a keratinocytes:fibroblastsratios of 1:49 to 1:1 the GM-CSF secretion passes 5000 pg/day. WhileGM-CSF secretion is highest at a keratinocyte:fibroblast ratio of 1:4,it is apparent that the largest increase in GM-CSF secretion is gainedwhen passing from a ration of 1:99 to 1:49.

Moreover, it is also evident that VEGF production is highly dependant onthe ratio of keratinocytes to fibroblasts in component #2. In theexperiments detailed in FIGS. 5 a and 5 b, there appears to be optimalsecretion of VEGF near a keratinocytes:fibroblasts ratio of 1:8, sincefurther increasing the number of keratinocytes leads to a decrease inthe quantity of VEGF secreted.

At different cell ratios, the application of a greater number of spraysalso leads to an increase in growth factor secretion levels. Data isshown as average±SEM (n=4, 1 spray; n=3, 2 sprays).

Example 10 Comparison of Storage at −160° C. Versus −80° C. During OneWeek

FIG. 7 shows a comparison of growth factor secretion by cells storedcryopreserved at −160° C. versus at −80° C. for a period of one week.The cryoprotectant used in this experiment was a 10% glycerol solutionwith 10% thrombin (Tisseel, Baxter) and the keratinocyte:fibroblastratio employed was 1:1. Prior to cryopreservation, cells were detachedfrom their culture surfaces using trypsin and subsequently irradiatedusing gamma (γ) irradiation at 80Gy. A controlled rate freezer was usedto gradually cool cell preparations to −80° C. After thawing one weeklater, one spray (130 μl) of the cell preparation (1.3 million cells at10 million cells/ml) were spray mixed with one spray (130 μl) offibrinogen in single wells of a 24 well petri dish. The results showGM-CSF, VEGF, and bFGF secretion during day 2 for three samples storedfor one week at −160° C. and four samples stored for one week at −80° C.compared to a control fresh (unfrozen) sample containing the samecryoprotectant. Secreted GM-CSF and VEGF were dosed in the culturemedia, while bFGF was dosed in the fibrin matrix. Secretion dataindicates that both −80° C. and −160° C. are suitable for storage,though −160° C. may be preferable when using a 10% glycerol solution.Data is shown as average±SEM (n=4).

Example 11 Comparison of Secretion after One Week Storage at −80° C. in10% Glycerol Versus 15% Glycerol

FIG. 8 shows a comparison of GM-CSF, VEGF and bFGF secretion by cellscryopreserved at −80° C. in 10% glycerol versus in 15% glycerol for aperiod of one week. The keratinocyte:fibroblast ratio used in thisexample was 1:1 mixed with 10% thrombin (Tisseel, Baxter) and acryoprotectant. Prior to cryopreservation, cells were detached fromtheir culture surfaces using trypsin and subsequently irradiated usinggamma (γ) irradiation at 80Gy. A controlled rate freezer was used togradually cool cell preparations to −80° C. After thawing one weeklater, one spray (130 μl) of the cell preparation (1.3 million cells at10 million cells/ml) were spray mixed with one spray (130 μl) offibrinogen in single wells of a 24 well petri dish.

The results show GM-CSF, VEGF, and bFGF secretion during day 2 for foursamples stored for one week at −80° C. using 10% glycerol and foursamples stored for one week at −80° C. using 15% glycerol compared tocontrol fresh (unfrozen) samples containing the same cryoprotectantconcentrations. A trend of higher protein secretion was observed insamples stored in 15% glycerol versus those kept in 10% glycerol.Secreted GM-CSF and VEGF were dosed in the culture media while bFGF wasdosed in the fibrin matrix. Data is presented as average±SEM (n=4).

Example 12 Comparison of Secretion After One Week Storage at −80° C. in15% Glycerol Versus 15% Glycerol+5% HSA

FIG. 9 shows a comparison of growth factor secretion by cellscryopreserved at −80° C. in 15% glycerol versus 15% glycerol+5% HumanSerum Albumin (HSA) (Griffols) for a period of one week. Thekeratinocyte:fibroblast ratio used in this example was 1:3 mixed with10% thrombin (Tisseel, Baxter) and a cryoprotectant. Prior tocryopreservation, cells were detached from their culture surfaces usingtrypsin and subsequently irradiated using gamma (γ) irradiation at 80Gy.A controlled rate freezer was used to gradually cool cell preparationsto −80° C. After thawing one week later, one spray (130 μl) of the cellpreparation (1.3 million cells at 10 million cells/ml) were spray mixedwith one spray (130 μl) of fibrinogen in single wells of a 24 well petridish. The results show GM-CSF, VEGF, and bFGF secretion during day 2 forthree samples stored for one week at −80° C. using 15% glycerol andthree samples stored for one week at −80° C. using 15% glycerol+5% HSA(Griffols) compared to control fresh (unfrozen) samples containing thesame cryoprotectants. Secretion data indicates that the addition ofHuman Serum Albumin improves the frozen product formulation bypermitting higher protein secretion levels. Data is presented asaverage±SEM (n=4).

Example 13 Bioactivity of Keratinocyte and Fibroblast Mixtures FollowingLong-Term Storage at −80° C.

FIG. 10 details the secretion of the human proteins GM-CSF and VEGF bycell preparations following storage at a temperature of −80° C. forextended periods. Data from three separate clinical production batchesis shown. Batches containing a ratio of human primary fibroblasts tokeratinocytes of 1:1 at a final concentration of 10×10⁶ cells/ml wereirradiated using gamma (γ) irradiation at 80Gy and frozen in a solutioncontaining thrombin, 15% glycerol and 5% human serum albumin. Samplesfrom each production batch were thawed following 1, 4, 8, and 12 weeksstorage at −80° C. Thawed samples were subsequently sprayed into 24-wellplates for testing. In individual wells, a single spray (130 μl) ofcells+thrombin+cryoprotectant is mixed with a single spray (130 μl) offibrinogen. The mixture of these two sprays creates a fibrin polymermatrix containing living fibroblasts and keratinocytes. The secretion ofproteins by cells trapped in the fibrin matrix is measured during day 2(the period lasting from 24 hours to 48 hours after thawing).

Secretion of GM-CSF and VEGF into the media by thawed cell preparationsremains relatively stable over a period of storage lasting 3 months at−80° C. Slight variations in GM-CSF secretion was seen from individualbatch to batch, though secretion within a batch appeared to berelatively stable. Secreted VEGF appeared stable, on average, both froma batch to batch perspective as well as within individual productionbatches. This data reveals that, with respect to GM-CSF and VEGFsecretion, the product is stable in a cryogenic state at −80° C. forperiods lasting at least 12 weeks. Data is presented as average±SEM.

Example 14 Comparison of Secretion After One Week Storage at −80° C. forDifferent Keratinocyte:Fibroblast Ratios

FIG. 11 shows the secretion of growth factors from cryopreserved cellpreparation formulations following one week of storage at −80° C. Thegraph shows differences in secretion for various human primarykeratinocyte:fibroblast ratios, including 1:0, 1:1, and 1:9 as well asdifferences associated with total cell concentrations of 5, 10 and 20million cells/ml. Prior to cryopreservation, cells were detached fromtheir culture surfaces using trypsin and subsequently irradiated usinggamma (γ) irradiation at 80Gy. A controlled rate freezer was used togradually cool cell preparations to −80° C. After thawing one weeklater, one spray (130 μl) of the cell preparation (5, 10 and 20 millioncells/ml) were spray mixed with one spray (130 μl) of fibrinogen insingle wells of a 24 well petri dish.

A comparison of the secretion data for the 1:0 ratio and the 1:1 ratioshowed the importance of adding fibroblasts to the keratinocytes interms of GM-CSF and VEGF production. In all cases, there was a cell-dosedependant relationship with the secretion of the growth factors duringthe second day after thawing. The data also demonstrated that reducingthe number of keratinocytes (i.e. by reducing thekeratinocyte:fibroblast ratio to 1:9) did not lead to a reduction inoverall growth factor secretion. These results were in accordance withthe data obtained for “fresh” (non-frozen) cell preparations (seeExample 9, supra) and suggested that only minimal quantities ofkeratinocytes were needed to produce the synergistic effect achieved bymixing the keratinocytes and fibroblasts together. This study alsodemonstrated that fibroblasts play an important role in the productionof VEGF by cell preparations.

Example 15 Comparison of Growth Factor Secretion in Fresh andCryopreserved Samples Stored at −80° C.

Prior to cryopreservation, cells were detached from their culturesurfaces using trypsin and subsequently irradiated using gamma (γ)irradiation at 80Gy. Cell concentrations tested included 5, 10, and 20million cells/ml (with a keratinocyte:fibroblast ratio 1:1), each mixedwith 10% thrombin (Tisseel, Baxter) and the cryoprotectant. A controlledrate freezer was used to gradually cool cell preparations to −80° C.After thawing one week later, one spray (130 μl) of the cell preparation(5, 10 and 20 million cells/ml) were spray mixed with one spray (130 μl)of fibrinogen in single wells of a 24 well petri dish. To assessvariability in frozen (cryopreserved) samples, 3 separate tubes for eachcondition were cryopreserved for one week at −80° C. Upon thawing, fivesamples were made per tube. The data presented in Table 8 is the averageof the 15 samples available for each condition. The reproduciblesecretion data observed in the three frozen and thawed tubes percondition attests to the quality of the cryopreservation method. Forfresh preparations, either 3 or 4 samples were made. Secretion of GM-CSFand VEGF by both freshly trypsinized and frozen cell preparations wasobserved to increase as cell density in the fibrin matrix increased from2.5 to 5 to 10 million cells/ml (corresponding to 5, 10 and 20 millioncells/ml found in original frozen preparations). This furtherillustrates the potential of dosing therapeutic effects by cell-basedtreatments.

TABLE 8 GM-CSF (pg) VEGF (pg) Sample % % Sample Name No. Average SEMFresh Average SEM Fresh 5 million/ml, 4 40 14 100 1433 289 100 Fresh 10million/ml, 4 270 128 100 3418 1293 100 Fresh 20 million/ml, 3 751 16100 5628 1150 100 Fresh 5 million/ml, 15 81 11 202.5 986 96 68.8 1 weekat −80° C. 10 million/ml, 15 254 21 94.1 2135 254 62.5 1 week at −80° C.20 million/ml, 15 554 51 73.8 3462 335 61.5 1 week at −80° C.

Example 16 Evaluation of the Safety of the Frozen Cell Preparation ofthe Invention

The safety of the frozen wound-healing cell preparation of the inventionhas been evaluated in a multicenter, open phase I study. The cellpreparation consisted of a kit containing two components. Component #1was a suspension of fibrinogen and component #2 was a suspension ofkeratinocytes and fibroblasts mixed in thrombin and cryoprotectant. Cellpreparations were frozen at −80° C., shipped to the clinic site at −80°C. and stored at −80° C. on site until use. Immediately prior to use,the cell preparation was thawed in a heated water bath. After thawingthe two components (fibrinogen) and (cell+thrombin+cryoprotectants) weresequentially spray applied on the wound site, forming a thin fibrinmatrix containing living keratinocytes and fibroblasts. Fourteenpatients with chronic venous leg ulcers not responding to standardtreatment with dressings and compression for at least 4-weeks (run-inphase) were enrolled in 5 centers in the Netherlands and Dutch Antilles.Ulcer sizes at baseline ranged from 0.3 to 20.4 cm2 (mean 5.8).Concomitant to the standard treatment, the cell preparation was thenapplied once weekly for up to 12 weeks or until complete closure,whichever came first.

No serious adverse events were reported in relation to this cellpreparation. Four moderate to severe adverse events were thought to beattributable to the cell preparation (3× ulcer pain, 1× with increasingulcer size). Moreover, there were no clinical signs of wound infection.Complete closure at week 12 was observed in 10 patients, 7 within 4weeks and 3 within 4 to 12 weeks of treatment. Mean time to closure was5.4 weeks.

In conclusion, the cell preparation of the invention is safe and welltolerated for the treatment of chronic venous leg ulcers.

Example 17 Spray Applied Living Keratinocytes and Fibroblasts as aBiologically Active Wound Dressing

Primary fibroblasts and keratinocytes residing in the skin, naturallysecrete a cocktail of growth factors and cytokines that act to stimulatethe wound healing response following interruption of the cutaneousbarrier. To mimic this natural process, a living cell-based wounddressing was developed for the treatment of chronic venous ulcers. Itconsists of two components: 1) a solution of fibrinogen; and 2) asuspension of keratinocytes and fibroblasts in thrombin andcryoprotectant. The product is stored frozen at −80° C. until use, atwhich time it is thawed, with the two components applied sequentially tothe wound surface using a spray applicator. In this manner,polymerization of the fibrin occurs on the wound with delivered cellsbecoming trapped in a thin layer of fibrin at the ulcer site.

Mixtures of allogeneic, growth-arrested primary keratinocytes andfibroblasts were observed to secrete different levels of therapeuticproteins (VEGF, HGF, GM-CSF, bFGF, and KGF) depending on the ratiosemployed. Secretion of GM-CSF was dependant on the synergy derived fromthe mutual presence of fibroblasts and keratinocytes. Increasing thecell concentration in the final wound dressing from 1.25×10⁶ cells/ml to5×10⁶ cells/ml led to an elevated secretion of growth factors andcytokines. Preliminary studies have shown that cell preparations remainbiologically active for at least 2 months when stored at −80° C.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A composition comprising: (a) cells comprising keratinocytes orfibroblasts, or mixtures thereof, that secrete one or more biologicallyactive molecules selected from the group consisting of GM-CSF, VEGF,KGF, bFGF, TGFβ, angiopoietin, EGF, IL-Iβ, IL-6, IL-8, TGFα, and TNFα;(b) an extracellular matrix comprising alginate; (c) a bufferedsolution; and (d) human serum albumin or a cryoprotectant, wherein thecells are allogeneic and mitotically inactive.
 2. The composition ofclaim 1, wherein the cells are mitotically inactivated by irradiation.3. The composition of claim 1, wherein the composition compriseskeratinocytes and fibroblasts and the ratio of keratinocytes tofibroblasts is 1:9.
 4. The composition of claim 1, wherein thecomposition is a paste.
 5. The composition of claim 1, wherein thecomposition comprises human serum albumin and a cryoprotectant.
 6. Thecomposition of claim 1, wherein the buffered solution is HBSS.
 7. Thecomposition of claim 1, wherein the buffered solution comprises Ca⁺⁺. 8.The composition of claim 1, wherein the composition comprises humanserum albumin.
 9. The composition of claim 1, wherein the compositioncomprises a cryoprotectant.
 10. The composition of claim 9 wherein thecryoprotectant is glycerol.
 11. The composition of claim 9, wherein thecomposition is cyropreserved.
 12. The composition of claim 9, whereinthe composition is comprised in a sterile vial.